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The 4D Nucleome (4DN) Network aims to elucidate the complex structure and organization of chromosomes in the nucleus and the impact of their disruption in disease biology. We present the 4DN Data Portal (https://data.4dnucleome.org/), a repository for datasets generated in the 4DN network and relevant external datasets. Datasets were generated with a wide range of experiments, including chromosome conformation capture assays such as Hi-C and other innovative sequencing and microscopy-based assays probing chromosome architecture. All together, the 4DN data portal hosts more than 1800 experiment sets and 36000 files. Results of sequencing-based assays from different laboratories are uniformly processed and quality-controlled. The portal interface allows easy browsing, filtering, and bulk downloads, and the integrated HiGlass genome browser allows interactive visualization and comparison of multiple datasets. The 4DN data portal represents a primary resource for chromosome contact and other nuclear architecture data for the scientific community.

The 4D Nucleome (4DN) Network aims to elucidate the complex structure and organization of chromosomes in the nucleus and the impact of their disruption in disease biology. We present the 4DN Data Portal (https://data.4dnucleome.org/), a repository for datasets generated in the 4DN network and relevant external datasets. Datasets were generated with a wide range of experiments, including chromosome conformation capture assays such as Hi-C and other innovative sequencing and microscopy-based assays probing chromosome architecture. All together, the 4DN data portal hosts more than 1800 experiment sets and 34000 files. Results of sequencing-based assays from different laboratories are uniformly processed and quality-controlled. The portal interface allows easy browsing, filtering, and bulk downloads, and the integrated HiGlass genome browser allows interactive visualization and comparison of multiple datasets. The 4DN data portal represents a primary resource for chromosome contact and other nuclear architecture data for the scientific community.

Since 1992, FlyBase (flybase.org) has been an essential online resource for the Drosophila research community. Concentrating on the most extensively studied species, Drosophila melanogaster, FlyBase includes information on genes (molecular and genetic), transgenic constructs, phenotypes, genetic and physical interactions, and reagents such as stocks and cDNAs. Access to data is provided through a number of tools, reports, and bulk-data downloads. Looking to the future, FlyBase is expanding its focus to serve a broader scientific community. In this update, we describe new features, datasets, reagent collections, and data presentations that address this goal, including enhanced orthology data, Human Disease Model Reports, protein domain search and visualization, concise gene summaries, a portal for external resources, video tutorials and the FlyBase Community Advisory Group.

We report the current status of the FlyBase annotated gene set for Drosophila melanogaster and highlight improvements based on high-throughput data. The FlyBase annotated gene set consists entirely of manually annotated gene models, with the exception of some classes of small non-coding RNAs. All gene models have been reviewed using evidence from high-throughput datasets, primarily from the modENCODE project. These datasets include RNA-Seq coverage data, RNA-Seq junction data, transcription start site profiles, and translation stop-codon read-through predictions. New annotation guidelines were developed to take into account the use of the high-throughput data. We describe how this flood of new data was incorporated into thousands of new and revised annotations. FlyBase has adopted a philosophy of excluding low-confidence and low-frequency data from gene model annotations; we also do not attempt to represent all possible permutations for complex and modularly organized genes. This has allowed us to produce a high-confidence, manageable gene annotation dataset that is available at FlyBase (http://flybase.org). Interesting aspects of new annotations include new genes (coding, non-coding, and antisense), many genes with alternative transcripts with very long 3′ UTRs (up to 15–18 kb), and a stunning mismatch in the number of male-specific genes (approximately 13% of all annotated gene models) vs. female-specific genes (less than 1%). The number of identified pseudogenes and mutations in the sequenced strain also increased significantly. We discuss remaining challenges, for instance, identification of functional small polypeptides and detection of alternative translation starts.

In the context of the FlyBase annotated gene models in Drosophila melanogaster, we describe the many exceptional cases we have curated from the literature or identified in the course of FlyBase analysis. These range from atypical but common examples such as dicistronic and polycistronic transcripts, noncanonical splices, trans-spliced transcripts, noncanonical translation starts, and stop-codon readthroughs, to single exceptional cases such as ribosomal frameshifting and HAC1-type intron processing. In FlyBase, exceptional genes and transcripts are flagged with Sequence Ontology terms and/or standardized comments. Because some of the rule-benders create problems for handlers of high-throughput data, we discuss plans for flagging these cases in bulk data downloads.

We have sequenced the genome of a second Drosophila species, Drosophila pseudoobscura, and compared this to the genome sequence of Drosophila melanogaster, a primary model organism. Throughout evolution the vast majority of Drosophila genes have remained on the same chromosome arm, but within each arm gene order has been extensively reshuffled, leading to a minimum of 921 syntenic blocks shared between the species. A repetitive sequence is found in the D. pseudoobscura genome at many junctions between adjacent syntenic blocks. Analysis of this novel repetitive element family suggests that recombination between offset elements may have given rise to many paracentric inversions, thereby contributing to the shuffling of gene order in the D. pseudoobscura lineage. Based on sequence similarity and synteny, 10,516 putative orthologs have been identified as a core gene set conserved over 25–55 million years (Myr) since the pseudoobscura/melanogaster divergence. Genes expressed in the testes had higher amino acid sequence divergence than the genome-wide average, consistent with the rapid evolution of sex-specific proteins. Cis-regulatory sequences are more conserved than random and nearby sequences between the species—but the difference is slight, suggesting that the evolution of cis-regulatory elements is flexible. Overall, a pattern of repeat-mediated chromosomal rearrangement, and high coadaptation of both male genes and cis-regulatory sequences emerges as important themes of genome divergence between these species of Drosophila.

The lark gene was initially identified in a behavioral screen for flies that displayed circadian rhythm defects (Newby and Jackson, 1993, 1996). It encodes an RNAbinding protein of the RNA Recognition Motif (RRM) class. Whereas lark heterozygotes exhibit a rhythm defect, homozygotes die during embryonic development because of a zygotic requirement for the gene product. Subsequently, it was shown that there is also a maternal requirement for lark function during oogenesis and early embryonic development (McNeil et al., 1999). Currently, several fly labs are pursuing studies of LARK function as related to oogenesis and embryonic development. LARK protein contains three potential RNA binding domains, two RNA recognition motifs, and a single retroviral type zinc finger, that have been shown to be important for LARK function during development but appear to play functionally redundant roles in the circadian regulation of eclosion (Newby and Jackson, 1996; McNeil et al., 2000). The LARK protein is widely expressed in most if not all tissues and throughout development (Zhang et al., 2000). In the majority of cells that express LARK, the protein has a nuclear localization. A notable exception occurs in a subset of neurosecretory cells that express the neuropeptide crustacean cardioactive peptide (CCAP), in which LARK is cytoplasmic. Of interest for the rhythm phenotype, CCAP cells exhibit circadian changes in LARK abundance (Zhang et al., 2000). In order to further characterize LARK protein function we generated pP{UAS-lark T:Ivir }(UAS-lark-3HA, Fig. 1, panel a). To distinguish GAL4 driven expression of the UAS-lark transgene from endogenous LARK expression, sequences encoding a triple hemagglutinin (HA) epitope were incorporated into the construct immediately prior to the LARK stop codon. Seven independent UAS-lark3HA transgenic lines were generated and here we report the preliminary characterization of three of these lines. We employed one line carrying a second chromosome insertion (w; P{w mC UAS-lark }94A) and two lines with independent third chromosome insertions (w; P{w mC UAS-lark }9A and (w; P{w mC UAS-lark }23A). All three lines are homozygous viable and fertile. To examine the effect of LARK overexpression, we first used the strong ubiquitous Act5C-GAL4 driver to drive expression of UAS-lark-3HA transgenes. Flies carrying an Act5C-GAL4 third chromosome insert (balanced over TM6b, Tb) were crossed to flies homozygous for a UAS-lark-3HA transgene. Adults carrying both the Act5C-GAL4 driver and any of the three UAS-lark-3HA transgenes were never observed; i.e., the only adult progeny that resulted from these crosses carried the TM6b, Tb chromosome (Table 1). In the case of two of the UAS-lark-3HA lines, 94A and 9A, some of the LARK overexpressing flies were able to develop to the early pupal stage (as indicated by a few Tb pupae in the vials; data not shown). However, these individuals ceased to develop soon after pupal formation and no brown or black Tb pupae were observed in these crosses. In the case of crosses with the 23A UAS-lark-3HA line, Tb pupae were never observed, suggesting that ubiquitous overexpression of LARK from this insertion is lethal prior to pupation. To determine if the observed lethality was caused by increased LARK protein expression in the nervous system, we used an elav-GAL4 driver (c155) to express UAS-lark-3HA transgenes in all differentiated neurons. Such a pan-neural overexpression of LARK caused lethality, the severity of which was dependent on the particular UAS-lark-3HA line employed in crosses. For example, crosses of males bearing the X-linked elav-GAL4 driver to females homozygous for the 23A UAS-lark-3HA transgene failed to produce any female offspring (Table 1). In similar crosses with elav-GAL4 and either the 94A or 9A UAS-lark-3HA transgene, GAL4/UAS flies were observed to eclose at a reduced frequency compared to UAS sibling controls (Table 1). Of the GAL4/UAS flies that do eclose, all display a cuticle-tanning defect characterized by a significant delay in cuticle hardening and pigmentation (not seen in control flies). In addition, the GAL4/UAS flies do not show normal wing expansion (Fig. 1, panel b). With the 94A UAS-lark-3HA transgene, none of the GAL4/UAS flies ever expanded their wings. Approximately 2% of the GAL4/UAS flies displayed nor

Mental retardation is a pervasive societal problem, 25 times more common than blindness for example. Fragile X syndrome, the most common form of inherited mental retardation, is caused by mutations in the FMR1 gene. Fragile X patients display neurite morphology defects in the brain, suggesting that this may be the basis of their mental retardation. Drosophila contains a single homolog of FMR1, dfxr (also called dfmr1). We analyzed the role of dfxr in neurite development in three distinct neuronal classes. We find that DFXR is required for normal neurite extension, guidance, and branching. dfxr mutants also display strong eclosion failure and circadian rhythm defects. Interestingly, distinct neuronal cell types show different phenotypes, suggesting that dfxr differentially regulates diverse targets in the brain.

Release 6, the latest reference genome assembly of the fruit fly Drosophila melanogaster, was released by the Berkeley Drosophila Genome Project in 2014; it replaces their previous Release 5 genome assembly, which had been the reference genome assembly for over 7 years. With the enormous amount of information now attached to the D. melanogaster genome in public repositories and individual laboratories, the replacement of the previous assembly by the new one is a major event requiring careful migration of annotations and genome-anchored data to the new, improved assembly. In this report, we describe the attributes of the new Release 6 reference genome assembly, the migration of FlyBase genome annotations to this new assembly, how genome features on this new assembly can be viewed in FlyBase (http://flybase.org) and how users can convert coordinates for their own data to the corresponding Release 6 coordinates.

Genetic analysis in Drosophila melanogaster has identified molecules important for the function of insect circadian clocks, and this has resulted in the elaboration of explicit biochemical models of the clock mechanism. Comparable molecular genetic analysis coupled with neuroanatomical approaches has also delineated cellular elements of the circadian pacemaker controlling insect activity rhythms. However, not much is known about the transfer of temporal information from clock cells in the insect brain to downstream neural elements or other target cells that are regulated by the clock (i.e. clock output pathways). In this review, we focus on the insect literature, with special reference to the fruitfly D. melanogaster and the hawkmoth Manduca sexta, to discuss the candidate molecules, biochemical mechanisms and cell types implicated in the clock control of behavior.

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA-binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)-containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA-binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly-emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA-binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 14–29, 2000

Photic entrainment of insect circadian rhythms can occur through either extraretinal (brain) or retinal photoreceptors, which mediate sensitivity to blue light or longer wavelengths, respectively. Although visual transduction processes are well understood in the insect retina, almost nothing is known about the extraretinal blue light photoreceptor of insects. We now have identified and characterized a candidate blue light photoreceptor gene inDrosophila(DCry) that is homologous to the cryptochrome (Cry) genes of mammals and plants. TheDCrygene is located in region 91F of the third chromosome, an interval that does not contain other genes required for circadian rhythmicity. The protein encoded byDCryis ∼50% identical to the CRY1 and CRY2 proteins recently discovered in mammalian species. As expected for an extraretinal photoreceptor mediating circadian entrainment,DCrymRNA is expressed within the adult brain and can be detected within body tissues. Indeed, tissuein situhybridization demonstrates prominent expression in cells of the lateral brain, which are close to or coincident with theDrosophilaclock neurons. Interestingly,DCrymRNA abundance oscillates in a circadian manner inDrosophilahead RNA extracts, and the temporal phasing of the rhythm is similar to that documented for the mouseCry1mRNA, which is expressed in clock tissues. Finally, we show that changes inDCrygene dosage are associated predictably with alterations of the blue light resetting response for the circadian rhythm of adult locomotor activity.

By monitoring the mitotic transmission of a marked chromosome bearing a defective centromere, we have identified conditional alleles of two genes involved in chromosome segregation (cse). Mutations in CSE1 and CSE2 have a greater effect on the segregation of chromosomes carrying mutant centromeres than on the segregation of chromosomes with wild-type centromeres. In addition, the cse mutations cause predominantly nondisjunction rather than loss events but do not cause a detectable increase in mitotic recombination. At the restrictive temperature, cse1 and cse2 mutants accumulate large-budded cells, with a significant fraction exhibiting aberrant binucleate morphologies. We cloned the CSE1 and CSE2 genes by complementation of the cold-sensitive phenotypes. Physical and genetic mapping data indicate that CSE1 is linked to HAP2 on the left arm of chromosome VII and CSE2 is adjacent to PRP2 on chromosome XIV. CSE1 is essential and encodes a novel 109-kDa protein. CSE2 encodes a 17-kDa protein with a putative basic-region leucine zipper motif. Disruption of CSE2 causes chromosome missegregation, conditional lethality, and slow growth at the permissive temperature.

The availability of sequenced genomes from 12 Drosophila species has enabled the use of comparative genomics for the systematic discovery of functional elements conserved within this genus. We have developed quantitative metrics for the evolutionary signatures specific to protein-coding regions and applied them genome-wide, resulting in 1193 candidate new protein-coding exons in the D. melanogaster genome. We have reviewed these predictions by manual curation and validated a subset by directed cDNA screening and sequencing, revealing both new genes and new alternative splice forms of known genes. We also used these evolutionary signatures to evaluate existing gene annotations, resulting in the validation of 87% of genes lacking descriptive names and identifying 414 poorly conserved genes that are likely to be spurious predictions, noncoding, or species-specific genes. Furthermore, our methods suggest a variety of refinements to hundreds of existing gene models, such as modifications to translation start codons and exon splice boundaries. Finally, we performed directed genome-wide searches for unusual protein-coding structures, discovering 149 possible examples of stop codon readthrough, 125 new candidate ORFs of polycistronic mRNAs, and several candidate translational frameshifts. These results affect >10% of annotated fly genes and demonstrate the power of comparative genomics to enhance our understanding of genome organization, even in a model organism as intensively studied as Drosophila melanogaster.

Past studies have implicated the Drosophila LARK protein in the circadian control of adult eclosion behavior. LARK has a broad tissue pattern of distribution, and is pan-neuronal in the differentiated brain. In certain peptidergic neurons, LARK abundance changes in a circadian manner. However, the precise cellular requirement for LARK, with respect to circadian behavior, is still not known. To explore this issue, we employed the GAL4/UAS binary expression system to increase LARK abundance in defined neuronal cell types. Interestingly, LARK expression in Crustacean Cardioactive Peptide (CCAP) neurons caused an early-eclosion phenotype, whereas a similar perturbation in the Eclosion Hormone (EH) cells resulted in abnormally late peaks of eclosion. Surprisingly, LARK expression in Pigment Dispersing Factor (PDF)- or TIMELESS (TIM)-containing clock neurons caused behavioral arrhythmicity, even though clock protein cycling was found to be normal in these flies. Although the observed effects of LARK expression mirrored those seen with genetic ablation of the relevant peptidergic populations, there was no evidence of defective cell development or morphology. This suggests that an alteration of cell function rather than cell death is the cause of the aberrant phenotypes. Diminished PDF immunoreactivity in flies expressing LARK in the PDF neurons suggests that an effect on neuropeptide synthesis, transport, or release may contribute to the observed arrhythmicity. Importantly, the expression of LARK in several other cell populations did not have detectable effects on development, viability or behavior, indicating a specificity of action within certain cell types.

Insect growth and metamorphosis is punctuated by molts, during which a new cuticle is produced. Every molt culminates in ecdysis, the shedding of the remains of the old cuticle. Both the timing of ecdysis relative to the molt and the actual execution of this vital insect behavior are under peptidergic neuronal control. Based on studies in the moth, Manduca sexta, it has been postulated that the neuropeptide Crustacean cardioactive peptide (CCAP)plays a key role in the initiation of the ecdysis motor program. We have used Drosophila bearing targeted ablations of CCAP neurons (CCAP KO animals) to investigate the role of CCAP in the execution and circadian regulation of ecdysis. CCAP KO animals showed specific defects at ecdysis, yet the severity and nature of the defects varied at different developmental stages. The majority of CCAP KO animals died at the pupal stage from the failure of pupal ecdysis, whereas larval ecdysis and adult eclosion behaviors showed only subtle defects. Interestingly, the most severe failure seen at eclosion appeared to be in a function required for abdominal inflation, which could be cardioactive in nature. Although CCAP KO populations exhibited circadian eclosion rhythms, the daily distribution of eclosion events (i.e.,gating) was abnormal. Effects on the execution of ecdysis and its circadian regulation indicate that CCAP is a key regulator of the behavior. Nevertheless, an unexpected finding of this work is that the primary functions of CCAP as well as its importance in the control of ecdysis behaviors may change during the postembryonic development of Drosophila.

Background: The recent completion of the Drosophila melanogaster genomic sequence to high quality and the availability of a greatly expanded set of Drosophila cDNA sequences, aligning to 78% of the predicted euchromatic genes, afforded FlyBase the opportunity to significantly improve genomic annotations. We made the annotation process more rigorous by inspecting each gene visually, utilizing a comprehensive set of curation rules, requiring traceable evidence for each gene model, and comparing each predicted peptide to SWISS-PROT and TrEMBL sequences. Results: Although the number of predicted protein-coding genes in Drosophila remains essentially unchanged, the revised annotation significantly improves gene models, resulting in structural changes to 85% of the transcripts and 45% of the predicted proteins. We annotated transposable elements and non-protein-coding RNAs as new features, and extended the annotation of untranslated (UTR) sequences and alternative transcripts to include more than 70% and 20% of genes, respectively. Finally, cDNA sequence provided evidence for dicistronic transcripts, neighboring genes with overlapping UTRs on the same DNA sequence strand, alternatively spliced genes that encode distinct, non-overlapping peptides, and numerous nested genes. Conclusions: Identification of so many unusual gene models not only suggests that some mechanisms for gene regulation are more prevalent than previously believed, but also underscores the complex challenges of eukaryotic gene prediction. At present, experimental data and human curation remain essential to generate high-quality genome annotations.