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Significance Apoptotic processes play an important role in the development and physiology of almost all metazoan clades. In the highly diverse group of insects, apoptotic pathways have been characterized in only a few dipteran and lepidopteran species, which may not be representative of all insect species. Here, we report the first complete annotation of the apoptotic pathway in a hemipteran insect, the pea aphid Acyrthosiphon pisum. We showed that its apoptotic pathway is rewired compared to other insects, with a significant increase in the number of inhibitors of apoptosis (IAPs) and evidence for functional diversification and structural modularity of this protein family. These novelties are widespread in the aphid lineage, suggesting a yet not understood novel aphid-specific function of IAPs., Apoptosis, a conserved form of programmed cell death, shows interspecies differences that may reflect evolutionary diversification and adaptation, a notion that remains largely untested. Among insects, the most speciose animal group, the apoptotic pathway has only been fully characterized in Drosophila melanogaster, and apoptosis-related proteins have been studied in a few other dipteran and lepidopteran species. Here, we studied the apoptotic pathway in the aphid Acyrthosiphon pisum, an insect pest belonging to the Hemiptera, an earlier-diverging and distantly related order. We combined phylogenetic analyses and conserved domain identification to annotate the apoptotic pathway in A. pisum and found low caspase diversity and a large expansion of its inhibitory part, with 28 inhibitors of apoptosis (IAPs). We analyzed the spatiotemporal expression of a selected set of pea aphid IAPs and showed that they are differentially expressed in different life stages and tissues, suggesting functional diversification. Five IAPs are specifically induced in bacteriocytes, the specialized cells housing symbiotic bacteria, during their cell death. We demonstrated the antiapoptotic role of these five IAPs using heterologous expression in a tractable in vivo model, the Drosophila melanogaster developing eye. Interestingly, IAPs with the strongest antiapoptotic potential contain two BIR and two RING domains, a domain association that has not been observed in any other species. We finally analyzed all available aphid genomes and found that they all show large IAP expansion, with new combinations of protein domains, suggestive of evolutionarily novel aphid-specific functions.

SUMMARY Combinatorial interactions among transcription factors (TFs) play essential roles in generating gene expression specificity and diversity in metazoans. Using yeast 2-hybrid (Y2H) assays on nearly all sequence-specific Drosophila TFs, we identified 1,983 protein-protein interactions (PPIs), more than doubling the number of currently known PPIs among Drosophila TFs. For quality assessment, we validated a subset of our interactions using MITOMI and bimolecular fluorescence complementation assays. We combined our interactome with prior PPI data to generate an integrated Drosophila TF-TF binary interaction network. Our analysis of ChIP-seq data, integrating PPI and gene expression information, uncovered different modes by which interacting TFs are recruited to DNA. We further demonstrate the utility of our Drosophila interactome in shedding light on human TF-TF interactions. This study reveals how TFs interact to bind regulatory elements in vivo and serves as a resource of Drosophila TF-TF binary PPIs for understanding tissue-specific gene regulation., Graphical Abstract, In Brief Combinatorial regulation by transcription factors (TFs) is one mechanism for achieving condition and tissue-specific gene regulation. Shokri et al. mapped TF-TF interactions between most Drosophila TFs, reporting a comprehensive TF-TF network integrated with previously known interactions. They used this network to discern distinct TF-DNA binding modes.

Mapping gene regulatory networks is a significant challenge in systems biology, yet only a few methods are currently capable of systems-level identification of transcription factors (TFs) that bind a specific regulatory element. We developed a microfluidic method for integrated systems-level interaction mapping of TF-DNA interactions, generating and interrogating an array of 423 full-length Drosophila TFs. With integrated systems-level interaction mapping, it is now possible to rapidly and quantitatively map gene regulatory networks of higher eukaryotes.

Two major families of nutritional proteins exist in insects, namely the vitellogenins and the yolk proteins. While in other insects only vitellogenins are found, cyclorraphan flies only contain yolk proteins. Possible sites of yolk protein synthesis are the fat body and the follicle cells surrounding the oocyte. We report the cloning of the yolk protein of the tsetse fly Glossina morsitans morsitans, a species with adenotrophic viviparity. The tsetse fly yolk protein could be aligned with other dipteran yolk proteins and with some vertebrate lipases. In contrast to the situation in most fly species, only a single yolk protein gene was found in the tsetse fly. Northern blot analysis showed that only the ovarian follicle cells, and not the fat body represents the site of yolk protein synthesis.

Understanding the relationship between genetic and phenotypic variation is one of the great outstanding challenges in biology. To meet this challenge, comprehensive genomic variation maps of human as well as of model organism populations are required. Here, we present a nucleotide resolution catalog of single-nucleotide, multi-nucleotide, and structural variants in 39 Drosophila melanogaster Genetic Reference Panel inbred lines. Using an integrative, local assembly-based approach for variant discovery, we identify more than 3.6 million distinct variants, among which were more than 800,000 unique insertions, deletions (indels), and complex variants (1 to 6,000 bp). While the SNP density is higher near other variants, we find that variants themselves are not mutagenic, nor are regions with high variant density particularly mutation-prone. Rather, our data suggest that the elevated SNP density around variants is mainly due to population-level processes. We also provide insights into the regulatory architecture of gene expression variation in adult flies by mapping cis-expression quantitative trait loci (cis-eQTLs) for more than 2,000 genes. Indels comprise around 10% of all cis-eQTLs and show larger effects than SNP cis-eQTLs. In addition, we identified two-fold more gene associations in males as compared to females and found that most cis-eQTLs are sex-specific, revealing a partial decoupling of the genomic architecture between the sexes as well as the importance of genetic factors in mediating sex-biased gene expression. Finally, we performed RNA-seq-based allelic expression imbalance analyses in the offspring of crosses between sequenced lines, which revealed that the majority of strong cis-eQTLs can be validated in heterozygous individuals., Author Summary One of the principal challenges in current biology is to understand the relationship between genetic and phenotypic variation. The increasing availability of genomic variation maps of human as well as of model organism populations (mouse and Arabidopsis) constitutes an important step towards meeting this challenge. However, despite its excellent track record as a premier model to understand genome function, no genome-wide variation data beyond single-nucleotide variants and microsatellites are currently available for D. melanogaster. Here, we present a comprehensive, nucleotide-resolution catalogue of variants of various types (single-nucleotide, multi-nucleotide, and structural variants) for 39 wild-derived inbred D. melanogaster lines based on high-throughput sequencing. This catalogue confirms that non–SNP variants account for more than half of genomic variation, allowing us to provide new insights into the non-random distribution of variants in the Drosophila genome. We further present genome-wide cis-associations with gene expression based on whole adult fly microarray data, revealing significant associations for about 2,000 genes. Most associations are sex-specific, providing evidence for a decoupling of the genomic, regulatory architecture between males and females.

Endothelin-converting enzyme is the key enzyme in the process of endothelin production. Endothelin is a peptide that plays an important role in vasoconstriction and the development of neural crest-derived cells in vertebrates. Activity assays performed on membrane extracts from Locusta migratoria brain revealed the existence of a protease activity responsible for the formation of mature endothelin-1 from its precursor, big endothelin. Cloning experiments led to a cDNA sequence (Lom ECE) with an open reading frame of 727 amino acid residues displaying all the characteristic ECE features. A comparison of ECE activity levels among different tissues of the locust showed a high enzyme activity in the gonads and midgut. RT-PCR experiments showed a wide tissue distribution of Lom ECE mRNA, with transcription being most abundant in brain tissue.

Angiotensin-converting enzyme, a member of the M2 metalloprotease family, and endothelin-converting enzyme, a member of the M13 family, are key components in the regulation of blood pressure and electrolyte balance in mammals. From this point of view, they serve as important drug targets. Recently, the involvement of these enzymes in the development of Alzheimer's disease was discovered. The existence of homologs of these enzymes in invertebrates indicates that these enzyme systems are highly conserved during evolution. Most invertebrates lack a closed circulatory system, which excludes the need for blood pressure regulators. Therefore, these organisms represent excellent targets for gaining new insights and revealing additional physiological roles of these important enzymes. This chapter reviews the structural and functional aspects of ACE and ECE and will particularly focus on these enzyme homologues in invertebrates.

An HPLC analysis of hemolymph extracts was undertaken to uncover differences between desert locusts, Schistocerca gregaria , reared under either crowded or isolated conditions. Some differences in the chromatographic pattern could be detected. One of the major peaks in the hemolymph of crowd-reared adults was found to be a minor one in isolated-reared individuals, whereas other peaks increased after solitarization. The differences became even more pronounced after several generations of isolated rearing. The dominant chromatographic peak in hemolymph extracts of the crowd-reared animals was identified as a novel peptide with a molecular mass of 6080 Da. Edman degradation in combination with enzymatic fragmentation and quadrupole-time of flight (Q-Tof) mass spectrometry revealed the full sequence: DNADEDTICVAADNKFYLYANSLKLYTCYNQLPKVYVVKPKSQCRSSLSDCPTS. This 54 aa-peptide is very abundant in hemolymph of crowd-reared adults. Its concentration in hemolymph amounts to 0.1 mM. To uncover the function, its effects were investigated in several bioassays, so far without positive results. One of the other peaks differentially expressed in the individuals of the two phases was identified as SGPI-2 (MW=3794 Da), which is a serine protease inhibitor in locusts.

A strong and constitutive angiotensin converting enzyme- or ACE-like activity was demonstrated in the hemolymph of the adult grey fleshfly Neobellieria bullata. In a competition assay, the N. bullata trypsin modulating oostatic factor (Neb-TMOF) was confirmed to be an in vitro substrate for this circulating Neb-ACE. Oral uptake of captopril, a selective and specific inhibitor of ACE, resulted in a complete phenotypic knockout of circulating ACE activity. When compared with control animals, captopril-fed female flies showed an increase in the liver meal–induced trypsin peak in the midgut and elevated levels of protein meal–induced yolk polypeptides in the hemolymph. The latter effect was not due to a slower vitellogenin uptake by the ovaries, because oocyte growth was not affected by the captopril treatment. The apparent synergism between the demonstrated ACE functionality and the previously reported effects of the oostatic peptide Neb-TMOF are discussed in the context of our recent finding that Neb-TMOF represents a prime candidate for being the first known in vivo substrate for circulating insect ACE. Arch. Insect Biochem. Physiol. 47:161–167, 2001. © 2001 Wiley-Liss, Inc.

Angiotensin converting enzyme (ACE) activity, defined as a captopril-inhibitable dipeptidyl carboxypeptidase activity towards 3H-hippurylglycylglycine, was demonstrated in haemolymph, testes and ovaries of the grey fleshfly Neobellieria bullata, hereby suggesting a physiological role for ACE in these particular tissues. While the ACE activity in haemolymph and testes reached relatively high levels, only minute ACE activity could be detected in ovaries throughout the entire vitellogenic cycle. Ovarian extracts of Neobellieria bullata do contain, however, in addition to Neb-TMOF, the Neobellieria bullata trypsin modulating oostatic factor which is an in vitro and a putative in vivo substrate of ACE in circulation, several other heat-stable molecules which individually function either as an ACE substrate or ACE inhibitor. Presumably these ACE interactive factors mask ACE activity in the fly ovaries, as measured by a classic substrate-binding assay. Purification and characterisation of these ACE substrates/inhibitors is in progress and is likely to facilitate the elucidation of the enigmatic physiological relevance of ACE in insects.

Drosophila melanogaster has one of the best characterized metazoan genomes in terms of functionally annotated regulatory elements. To explore how these elements contribute to gene regulation in the context of gene regulatory networks, we need convenient tools to identify the proteins that bind to them. Here, we present the development and validation of a highly automated protein-DNA interaction detection method, enabling the high-throughput yeast one-hybrid-based screening of DNA elements versus an array of full-length, sequence-verified clones containing 647 (over 85%) of predicted Drosophila transcription factors (TFs). Using six well-characterized regulatory elements (82 bp – 1kb), we identified 33 TF-DNA interactions of which 27 are novel. To simultaneously validate these interactions and locate their binding sites of involved TFs, we implemented a novel microfluidics-based approach that enables us to conduct hundreds of gel shift-like assays at once, thus allowing the retrieval of DNA occupancy data for each TF throughout the respective target DNA elements. Finally, we biologically validate several interactions and specifically identify two novel regulators of sine oculis gene expression and hence eye development.

Lithium and valproate (VPA) are drugs used in the management of bipolar disorder. Even though they reportedly act on various pathways, the transcriptional targets relevant for disease mechanism and therapeutic effect remain unclear. Furthermore, multiple studies used lymphoblasts of bipolar patients as a cellular proxy, but it remains unclear whether peripheral cells provide a good readout for the effects of these drugs in the brain. We used Drosophila culture cells and adult flies to analyze the transcriptional effects of lithium and VPA and define mechanistic pathways. Transcriptional profiles were determined for Drosophila S2-cells and adult fly heads following lithium or VPA treatment. Gene ontology categories were identified using the DAVID functional annotation tool with a cut-off of p < 0.05. Significantly enriched GO terms were clustered using REVIGO and DAVID functional annotation clustering. Significance of overlap between transcript lists was determined with a Fisher's exact hypergeometric test. Treatment of cultured cells and adult flies with lithium and VPA induces transcriptional responses in genes with similar ontology, with as most prominent immune response, neuronal development, neuronal function, and metabolism. (i) Transcriptional effects of lithium and VPA in Drosophila S2 cells and heads show significant overlap. (ii) The overlap between transcriptional alterations in peripheral versus neuronal cells at the single gene level is negligible, but at the gene ontology and pathway level considerable overlap can be found. (iii) Lithium and VPA act on evolutionarily conserved pathways in Drosophila and mammalian models.

Vitellogenins, cyclorraphan yolk proteins and lepidopteran minor yolk proteins are three classes of female-specific proteins that serve as an embryonic nutritional store. Similarity to vertebrate lipid-binding proteins was established for vitellogenins and yolk proteins, vitellogenins being related to apolipoprotein B and yolk proteins to lipases. Recently, similarity between yolk proteins and minor yolk proteins was reported and it was suggested that yolk proteins are more related to minor yolk proteins than to vertebrate lipases. In this study, we cloned five additional yolk proteins from the grey fleshfly Neobellieria bullata, formerly known as Sarcophaga bullata. We used this sequence data, combined with sequence data retrieved from the NCBI protein database to evaluate the yolk protein-lipase and the yolk protein-minor yolk protein relationship. We found no similarity between yolk proteins and minor yolk proteins, but we showed that yolk proteins are related to a family of lipases containing vertebrate hepatic and pancreatic lipases while minor yolk proteins are related to a family of lipases containing vertebrate gastric and lingual lipases. The fact that three different classes of yolk storage proteins show similarity to three different classes of vertebrate lipid-binding proteins strongly suggests that this lipid-binding feature is important for insect yolk storage proteins.

A protocol to follow the processing of angiotensin I into angiotensin II by rabbit angiotensin-converting enzyme (ACE) and its inhibition by a novel natural antagonist, the leech osmoregulator factor (LORF) using capillary zonal electrophoresis is described. The experiment was carried out using the Beckman PACE system and steps were taken to determine (a) the migration profiles of angiotensin and its yielded peptides, (b) the minimal amount of angiotensin II detected, (c) the use of different electrolytes and (d) the concentration of inhibitor. We demonstrated that LORF (IPEPYVWD), a neuropeptide previously found in leech brain, is able to inhibit rabbit ACE with an IC(50) of 19.8 micro m. Interestingly, its cleavage product, IPEP exhibits an IC(50) of 11.5 micro m. A competition assay using p-benzoylglycylglycylglycine and insect ACE established that LORF and IPEP fragments are natural inhibitors for invertebrate ACE. Fifty-four percent of insect ACE activity is inhibited with 50 micro m IPEP and 35% inhibition with LORF (25 mm). Extending the peptide at both N- and C-terminus (GWEIPEPYVWDES) and the cleavage of IPEP in IP abolished the inhibitory activity of both peptides. Immunocytochemical data obtained with antisera raised against LORF and leech ACE showed a colocalization between the enzyme and its inhibitor in the same neurons. These results showed that capillary zonal electrophoresis is a useful technique for following enzymatic processes with small amounts of products and constitutes the first evidence of a natural ACE inhibitor in invertebrates.

By means of a tracer assay using a labeled synthetic angiotensin converting enzyme (ACE) substrate hippurylglycylglycine, we have detected high ACE activity in the testes of the African migratory locust, Locusta migratoria. Lower, but significant, ACE activity was observed in midgut and hemolymph. In a two-step purification procedure involving anion exchange and gel permeation chromatography, we have purified LomACE from the locust testes. The enzyme of approximately 80 kDa shows substantial amino-acid sequence homology with ACE from both vertebrate and invertebrate origin. The ACE identity of the purified enzyme was further confirmed by cDNA cloning of the Locusta ACE fragment, which, after in silico translation, revealed a mature protein of 623 amino acids with a large structural similarity to other known ACE proteins.

The presence of angiotensin converting enzyme (ACE) in insects has been reported many times, but numerous questions about the functional role of this enzyme in insects remain. Here we show by RT-PCR experiments that ACE has a wide tissue distribution in Locusta migratoria, suggesting diverse roles for this enzyme in the locust. Immune challenge through injection of bacterial lipopolysaccharides resulted in a tenfold increase of ACE gene transcripts in the hemocytes and is suggestive for a role of ACE in the cellular defense of the locust. However, phenotypic knockout experiments with the ACE inhibitor captopril showed that ACE is not essential for the efficient clearance of injected E. coli bacteria.

Vitellogenic ovaries of the gray fleshfly Neobellieria bullata contain a variety of unidentified substances that interact, either as a substrate or as an inhibitor, with angiotensin converting enzyme (ACE). We here report the isolation and characterization of the first ACE interactive compound hereof. This 1312.7 Da peptide with the sequence NKLKPSQWISL, is substrate to both insect and human ACE. It is a novel peptide that shows high sequence similarity to a sequence at the N-terminal part of dipteran yolk polypeptides (YPs). We propose to call it N. bullata ovary-derived ACE interactive factor or Neb-ODAIF. Both insect and human ACE hydrolyze Neb-ODAIF by sequentially cleaving off two C-terminal dipeptides. K(m) values of Neb-ODAIF and Neb-ODAIF(1-9) (NKLKPSQWI) for human somatic ACE (sACE) are 17 and 81 microM, respectively. Additionally, Neb-ODAIF(1-7) (NKLKPSQ) also interacts with sACE (K(m/i)=90 microM). These affinity-constants are in range with those of the physiological ACE substrates and suggest the importance of Neb-ODAIF and its cleavage products in the elucidation of the physiological role of insect ACE. Alternatively, they can serve as lead compounds in the development of new drugs against ACE-related diseases in humans.

Angiotensin converting enzyme (ACE) was already discovered in insects in 1994, but its physiological role is still enigmatic. We have addressed this problem by purifying four new ACE substrates from the ovaries of the grey fleshfly, Neobellieria bullata. Their primary structures were identified as NKLKPSQWISLSD (Neb-ODAIF-1(1-13)), NKLKPSQWI (Neb-ODAIF-1(1-9)), SLKPSNWLTPSE (Neb-ODAIF-2) and LEQIYHL. Database analysis showed significant homology with amino acid sequence stretches as present in the N-terminal part of several fly yolk proteins. An antiserum raised against Neb-ODAIF-1(1-9) immunostained one out of three yolk protein bands of SDS/PAGE-separated fly haemolymph and egg homogenate, thus confirming that these peptides originate from a yolk protein gene product. Kinetic analysis of these peptides and of the peptides Neb-ODAIF and Neb-ODAIF-1(1-7) with insect ACE and human ACE show both similar and unique properties for insect ACE as compared with human C-domain ACE.

Transcription factors (TFs) are key regulators of cell fate. The estimated 755 genes that encode DNA binding domain-containing proteins comprise ∼5% of all Drosophila genes. However, the majority has remained uncharacterized so far due to the lack of proper genetic tools. We generated 594 site-directed transgenic Drosophila lines that contain integrations of individual UAS-TF constructs to facilitate spatiotemporally controlled misexpression in vivo. All transgenes were expressed in the developing wing, and two-thirds induced specific phenotypic defects. In vivo knockdown of the same genes yielded a phenotype for 50%, with both methods indicating a great potential for misexpression to characterize novel functions in wing growth, patterning, and development. Thus, our UAS-TF library provides an important addition to the genetic toolbox of Drosophila research, enabling the identification of several novel wing development-related TFs. In parallel, we established the chromatin landscape of wing imaginal discs by ChIP-seq analyses of five chromatin marks and RNA Pol II. Subsequent clustering revealed six distinct chromatin states, with two clusters showing enrichment for both active and repressive marks. TFs that carry such “bivalent” chromatin are highly enriched for causing misexpression phenotypes in the wing, and analysis of existing expression data shows that these TFs tend to be differentially expressed across the wing disc. Thus, bivalently marked chromatin can be used as a marker for spatially regulated TFs that are functionally relevant in a developing tissue.

Members of the M13 class of metalloproteases have been implicated in diseases and in reproductive fitness. Nevertheless, their physiological role remains poorly understood. To obtain a tractable model with which to analyze this protein family's function, we characterized the gene family in Drosophila melanogaster and focused on reproductive phenotypes. The D. melanogaster genome contains 24 M13 class protease homologs, some of which are orthologs of human proteases, including neprilysin. Many are expressed in the reproductive tracts of either sex. Using RNAi we individually targeted the five Nep genes most closely related to vertebrate neprilysin, Nep1-5, to investigate their roles in reproduction. A reduction in Nep1, Nep2, or Nep4 expression in females reduced egg laying. Nep1 and Nep2 are required in the CNS and the spermathecae for wild-type fecundity. Females that are null for Nep2 also show defects as hosts of sperm competition as well as an increased rate of depletion for stored sperm. Furthermore, eggs laid by Nep2 mutant females are fertilized normally, but arrest early in embryonic development. In the male, only Nep1 was required to induce normal patterns of female egg laying. Reduction in the expression of Nep2-5 in the male did not cause any dramatic effects on reproductive fitness, which suggests that these genes are either nonessential for male fertility or perform redundant functions. Our results suggest that, consistent with the functions of neprilysins in mammals, these proteins are also required for reproduction in Drosophila, opening up this model system for further functional analysis of this protein class and their substrates. ispartof: Genetics vol:196 issue:3 pages:781-797 ispartof: location:United States status: published

In recent years, new techniques have spurred the discovery of cis-regulatory DNA elements. These stretches of noncoding DNA contain combinations of recognition sites to which transcription factors (TFs) bind, and in doing so, these TFs can activate or repress transcription. These protein-DNA interactions form the core of gene regulatory networks (GRNs) that are responsible for the differential gene expression that allow diversification of cell types, developmental programs, and responses to the environment. The yeast one-hybrid system is a genetic assay to identify direct binding of proteins to DNA elements of interest and is, therefore, instrumental in uncovering these GRNs.

Keywords: Copy-Number Polymorphism ; Drosophila-Melanogaster Reference EPFL-ARTICLE-149499doi:10.1038/nmeth.f.308View record in Web of Science Record created on 2010-06-24, modified on 2017-05-12

Pax6 genes encode evolutionarily highly conserved transcription factors that are required for eye and brain development. Despite the characterization of mutations in Pax6 homologs in a range of organisms, and despite functional studies, it remains unclear what the relative importance is of the various parts of the Pax6 protein. To address this, we have studied the Drosophila Pax6 homolog eyeless. Specifically, we have generated new eyeless alleles, each with single missense mutations in one of the four domains of the protein. We show that these alleles result in abnormal eye and brain development while maintaining the OK107 eyeless GAL4 activity from which they were derived. We performed in vivo functional rescue experiments by expressing in an eyeless-specific pattern Eyeless proteins in which either the paired domain, the homeodomain, or the C-terminal domain was deleted. Rescue of the eye and brain phenotypes was only observed when full-length Eyeless was expressed, while all deletion constructs failed to rescue. These data, along with the phenotypes observed in the four newly characterized eyeless alleles, demonstrate the requirement for an intact Eyeless protein for normal Drosophila eye and brain development. They also suggest that some endogenous functions may be obscured in ectopic expression experiments.

Insulin/insulin-like growth factor (IGF) signaling constitutes an evolutionarily conserved pathway that controls growth, energy homeostasis, and longevity. In Drosophila melanogaster , key components of this pathway are the insulin-like peptides (Dilps). The major source of Dilps is a cluster of large neurons in the brain, the insulin-producing cells (IPCs). The genetic control of IPC development and function is poorly understood. Here, we demonstrate that the Pax6 homolog Eyeless is required in the IPCs to control their differentiation and function. Loss of eyeless results in phenotypes associated with loss of insulin signaling, including decreased animal size and increased carbohydrate levels in larval hemolymph. We show that mutations in eyeless lead to defective differentiation and morphologically abnormal IPCs. We also demonstrate that Eyeless controls IPC function by the direct transcriptional control of one of the major Dilps, dilp5 . We propose that Eyeless has an evolutionarily conserved role in IPCs with remarkable similarities to the role of vertebrate Pax6 in β cells of the pancreas.

The arthropod compound eye is one of the three main types of eyes observed in the animal kingdom. Comparison of the eyes seen in Insecta, Crustacea, Myriapoda and Chelicerata reveals considerable variation in terms of overall cell number, cell positioning, and photoreceptor rhabdomeres, yet, molecular data suggest there may be unexpected similarities. We review here the role of Pax6 in eye development and evolution and the relationship of Pax6 with other retinal determination genes and signaling pathways. We then discuss how the study of changes in Pax6 primary structure, in the gene networks controlled by Pax6 and in the relationship of Pax6 with signaling pathways may contribute to our insight into the relative role of conserved molecular-genetic mechanisms and emergence of evolutionary novelty in shaping the ommatidial eyes seen in the Arthropoda.

Angiotensin-converting enzyme, a member of the M2 metalloprotease family, and endothelin-converting enzyme, a member of the M13 family, are key components in the regulation of blood pressure and electrolyte balance in mammals. From this point of view, they serve as important drug targets. Recently, the involvement of these enzymes in the development of Alzheimer's disease was discovered. The existence of homologs of these enzymes in invertebrates indicates that these enzyme systems are highly conserved during evolution. Most invertebrates lack a closed circulatory system, which excludes the need for blood pressure regulators. Therefore, these organisms represent excellent targets for gaining new insights and revealing additional physiological roles of these important enzymes. This chapter reviews the structural and functional aspects of ACE and ECE and will particularly focus on these enzyme homologues in invertebrates.

A protocol to follow the processing of angiotensin I into angiotensin II by rabbit angiotensin-converting enzyme (ACE) and its inhibition by a novel natural antagonist, the leech osmoregulator factor (LORF) using capillary zonal electrophoresis is described. The experiment was carried out using the Beckman PACE system and steps were taken to determine (a) the migration profiles of angiotensin and its yielded peptides, (b) the minimal amount of angiotensin II detected, (c) the use of different electrolytes and (d) the concentration of inhibitor. We demonstrated that LORF (IPEPYVWD), a neuropeptide previously found in leech brain, is able to inhibit rabbit ACE with an IC(50) of 19.8 micro m. Interestingly, its cleavage product, IPEP exhibits an IC(50) of 11.5 micro m. A competition assay using p-benzoylglycylglycylglycine and insect ACE established that LORF and IPEP fragments are natural inhibitors for invertebrate ACE. Fifty-four percent of insect ACE activity is inhibited with 50 micro m IPEP and 35% inhibition with LORF (25 mm). Extending the peptide at both N- and C-terminus (GWEIPEPYVWDES) and the cleavage of IPEP in IP abolished the inhibitory activity of both peptides. Immunocytochemical data obtained with antisera raised against LORF and leech ACE showed a colocalization between the enzyme and its inhibitor in the same neurons. These results showed that capillary zonal electrophoresis is a useful technique for following enzymatic processes with small amounts of products and constitutes the first evidence of a natural ACE inhibitor in invertebrates.

Research on the angiotensin-converting enzyme (ACE) in insects has substantially advanced during the recent decade. The cloning of this enzyme in many insect species, the determination of the 3D-structure and several molecular and physiological studies have contributed to the characterization of insect ACE as we know it today: a functional enzyme with a putative role in reproduction, development and defense. The discovery of the endothelin-converting enzyme in insects occurred more recently and cloning of the corresponding cDNA has been carried out in only one insect species so far. However, activity studies and analysis of insect genomes indicate that this enzyme is also widely distributed among insect species. Making hypotheses about its putative function would be preliminary, but its wide tissue distribution suggests a major and diverse biological role.

After translation, the AKH I and AKH II precursors form three dimeric constructs prior to further processing into the respective AKHs and three dimeric Adipokinetic Hormone Precursor Related Peptides or APRPs (two homodimers and one heterodimer). By capillary liquid chromatography-tandem mass spectrometry we demonstrate that the APRPs in Locusta migratoria are further processed to form two smaller neuropeptides: DAADFADPYSFL (residue 36 to 47 of the AKH I precursor) and YADPNADPMAFL (residue 34 to 45 of the AKH II precursor). The peptides are designated as Adipokinetic Hormone Joining Peptide 1 (AKH-JP I) and 2 (AKH-JP II) respectively. Within the AKH I and AKH II precursor molecules, the classic KK and RR processing sites separate the AKH-JPs from the AKH I and II respectively. At the carboxyterminus, both AKH-JP I and II are flanked by Tyr-Arg, a cleaving site not described before. Such an unusual cleavage site suggests the presence, in the corpora cardiaca, of specific convertases. The AKH-JP-II does not stimulate lipid release from the fat body nor does it stimulate glycogen phosphorylase activity, both key functions of AKH.

A combined cross-platform approach is presented to experimentally identify and characterize interactions between mouse transcription factors and regulatory elements at unprecedented resolution and throughput., We generated a mouse-specific transcription factor (TF) library consisting of 750 full-length sequence-verified open-reading frame clones. We used this resource to develop a cross-platform pipeline to experimentally characterize mammalian regulatory elements of interest for interacting TFs at unprecedented throughput and resolution. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers novel TF–DNA interactions that are relevant in vivo., The comprehensive mapping of gene promoters and enhancers has significantly improved our understanding of how the mammalian regulatory genome is organized. An important challenge is to elucidate how these regulatory elements contribute to gene expression by identifying their trans-regulatory inputs. Here, we present the generation of a mouse-specific transcription factor (TF) open-reading frame clone library and its implementation in yeast one-hybrid assays to enable large-scale protein–DNA interaction detection with mouse regulatory elements. Once specific interactions are identified, we then use a microfluidics-based method to validate and precisely map them within the respective DNA sequences. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers many novel TF–DNA interactions. In addition, we provide evidence that several of these novel interactions are relevant in vivo and aid in elucidating the regulatory architecture of enhancers.