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International audience; How the vast array of neuronal diversity is generated remains an unsolved problem. Here, we investigate how 29 morphologically distinct leg motoneurons are generated from a single stem cell in Drosophila. We identify 19 transcription factor (TF) codes expressed in immature motoneurons just before their morphological differentiation. Using genetic manipulations and a computational tool, we demonstrate that the TF codes are progressively established in immature motoneurons according to their birth order. Comparing RNA and protein expression patterns of multiple TFs reveals that post-transcriptional regulation plays an essential role in shaping these TF codes. Two RNA-binding proteins, Imp and Syp, expressed in opposing gradients in immature motoneurons, control the translation of multiple TFs. The varying sensitivity of TF mRNAs to the opposing gradients of Imp and Syp in immature motoneurons decrypts these gradients into distinct TF codes, establishing the connectome between motoneuron axons and their target muscles.

SUMMARYTemporal factors expressed sequentially in neural stem cells, such as RNA binding proteins (RBPs) or transcription factors (TFs), are key elements in the generation of neuronal diversity. The molecular mechanism underlying how the temporal identity of stem cells is decoded into their progeny to generate neuronal diversity is largely unknown. Here, we used genetic and new computational tools to study with precision the unique fates of the progeny of a stem cell producing 29 morphologically distinct leg motoneurons (MNs) in Drosophila. We identified 40 TFs expressed in this MN lineage, 15 of which are expressed in a combinatorial manner in immature MNs just before their morphological differentiation. By following TF expression patterns at an earlier developmental stages, we discovered 19 combinatorial codes of TFs that were progressively established in immature MNs as a function of their birth order. The comparison of the RNA and protein expression profiles of 6 TFs revealed that post-transcriptional regulation plays an essential role in shaping these TF codes. We found that the two known RBPs, Imp and Syp, expressed sequentially in neuronal stem cells, are upstream regulators of the TF codes. Both RBPs are key players in the construction of axon-muscle connectome through the post-transcriptional regulation of 5 of the 6 TFs examined. By deciphering the function of Imp in the immature MNs with respect to the stem cell of the same lineage, we propose a model where RBPs shape the morphological fates of MNs through post-transcriptional regulation of TF codes in immature MNs. Taken together, our study reveals that immature MNs are plastic cells that have the potential to acquire many morphological fates. The molecular basis of MN plasticity originates in the broad expression of different TF mRNA, that are post-transcriptionally shaped into TF codes by Imp and Syp, and potentially by other RBPs that remain to be discovered, to determine their morphological fates.

Introduction Lipidomic profiling allows 100s if not 1000s of lipids in a sample to be detected and quantified. Modern lipidomics techniques are ultra-sensitive assays that enable the discovery of novel biomarkers in a variety of fields and provide new insight in mechanistic investigations. Despite much progress in lipidomics, there remains, as for all high throughput “omics” strategies, the need to develop strategies to standardize and integrate quality control into studies in order to enhance robustness, reproducibility, and usability of studies within specific fields and beyond. Objectives We aimed to understand how much results from lipid profiling in the model organism Caenorhabditis elegans are influenced by different culture conditions in different laboratories. Methods In this work we have undertaken an inter-laboratory study, comparing the lipid profiles of N2 wild type C. elegans and daf-2(e1370) mutants lacking a functional insulin receptor. Sample were collected from worms grown in four separate laboratories under standardized growth conditions. We used an UPLC-UHR-ToF–MS system allowing chromatographic separation before MS analysis. Results We found common qualitative changes in several marker lipids in samples from the individual laboratories. On the other hand, even in this controlled experimental system, the exact fold-changes for each marker varied between laboratories. Conclusion Our results thus reveal a serious limitation to the reproducibility of current lipid profiling experiments and reveal challenges to the integration of such data from different laboratories.

International audience; Ageing is accompanied by important chemical deregulations resulting in bodily metal imbalances. The way and extent to which these deregulations are associated with ageing processes are however poorly understood and their use as potential biomarkers of ageing has not been investigated. In this study, we report whole-body elementary concentrations and copper and zinc isotopic compositions of Caenorhabditis elegans in ageing wild type (i.e. normal'-lived) and mutant (i.e. short and long-lived) strains. We show that the strains are characterized by different levels of mutation-related variations such as in phosphorus and magnesium as well as in zinc isotopic composition. During ageing, strains are affected by elemental age-related variations, such as an increase in calcium and iron concentrations and a decrease in the copper isotopic composition and concentration for long-lived mutants. The deregulated metabolism of copper seems to be connected to ageing probably in association with the production of reactive oxygen species. We emphasize that the copper stable isotope composition could serve as a biomarker of normal or accelerated ageing in the future.

Motile cilia play important functions in many organisms. In Drosophila, hemingway (hmw) encodes a novel protein conserved in species with motile cilia. hmw-mutant flies are hearing impaired and male sterile. HMW is required for acquisition of motile properties of cilia in the fly sound receiver and stability of the sperm axoneme., Cilia play major functions in physiology and development, and ciliary dysfunctions are responsible for several diseases in humans called ciliopathies. Cilia motility is required for cell and fluid propulsion in organisms. In humans, cilia motility deficiencies lead to primary ciliary dyskinesia, with upper-airways recurrent infections, left–right asymmetry perturbations, and fertility defects. In Drosophila, we identified hemingway (hmw) as a novel component required for motile cilia function. hmw encodes a 604–amino acid protein characterized by a highly conserved coiled-coil domain also found in the human orthologue, KIAA1430. We show that HMW is conserved in species with motile cilia and that, in Drosophila, hmw is expressed in ciliated sensory neurons and spermatozoa. We created hmw-knockout flies and found that they are hearing impaired and male sterile. hmw is implicated in the motility of ciliated auditory sensory neurons and, in the testis, is required for elongation and maintenance of sperm flagella. Because HMW is absent from mature flagella, we propose that HMW is not a structural component of the motile axoneme but is required for proper acquisition of motile properties. This identifies HMW as a novel, evolutionarily conserved component necessary for motile cilium function and flagella assembly.

In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C. Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2). Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes. The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212–mus219). Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes. The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314–mus327). We have initiated efforts to identify these genes at the molecular level and report here the first two identified. The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene. We have also identified a second-chromosome mutant, mus215ZIII-2059, that uniformly reduces the frequency of meiotic recombination to

ATM/ATR kinases act as signal transducers in eukaryotic DNA damage and replication checkpoints. Mutations in ATM/ATR homologs have pleiotropic effects that range from sterility to increased killing by genotoxins in humans, mice, and Drosophila. Here we report the generation of a null allele of mei-41, Drosophila ATM/ATR homolog, and the use of it to document a semidominant effect on a larval mitotic checkpoint and methyl methanesulfonate (MMS) sensitivity. We also tested the role of mei-41 in a recently characterized checkpoint that delays metaphase/anaphase transition after DNA damage in cellular embryos. We then compare five existing mei-41 alleles to the null with respect to known phenotypes (female sterility, cell cycle checkpoints, and MMS resistance). We find that not all phenotypes are affected equally by each allele, i.e., the functions of MEI-41 in ensuring fertility, cell cycle regulation, and resistance to genotoxins are genetically separable. We propose that MEI-41 acts not in a single rigid signal transduction pathway, but in multiple molecular contexts to carry out its many functions. Sequence analysis identified mutations, which, for most alleles, fall in the poorly characterized region outside the kinase domain; this allowed us to tentatively identify additional functional domains of MEI-41 that could be subjected to future structure-function studies of this key molecule.

International audience; Ciliated neurons play an important role in sensory perception in many animals. Modified cilia at dendrite endings serve as sites of sensory signal capture and transduction. We describe Drosophila mutations that affect the transcription factor RFX and genetic rescue experiments that demonstrate its central role in sensory cilium differentiation. Rfx mutant flies show defects in chemosensory and mechanosensory behaviors but have normal phototaxis, consistent with Rfx expression in ciliated sensory neurons and neuronal precursors but not in photoreceptors. The mutant behavioral phenotypes are correlated with abnormal function and structure of neuronal cilia, as shown by the loss of sensory transduction and by defects in ciliary morphology and ultrastructure. These results identify Rfx as an essential regulator of ciliated sensory neuron differentiation in Drosophila.

This paper reports on a new role for mei-41 in cell cycle control during meiosis. This function is revealed by the requirement of mei-41 for the precocious anaphase observed in crossover-defective mutants. Normally in Drosophila oocytes, tension on the meiotic spindle causes a metaphase I arrest. This tension results because crossovers, and the resulting chiasmata, hold homologs together that are being pulled by kinetochore microtobules toward opposite spindle poles. In the absence of tension, such as in a recombination-defective mutant, metaphase arrest is not observed and meiosis proceeds through the two divisions. Here we show that in some recombination-defective mutants, the precocious anaphase requires the mei-41 gene product. For example, metaphase arrest is not observed in mei-218 mutants because of the severe reduction in crossing over. In mei-41 mei-218 double mutants, however, metaphase arrest was restored. The effect of mei-41 is dependent on double-strand break formation. Thus, in mutants that fail to initiate meiotic recombination the absence of mei-41 has no effect.

In contrast to vertebrate CBY, which functions in WNT signaling, Drosophila CBY is essential for normal basal body structure and function but dispensable for Wg signaling., Centriole-to–basal body conversion, a complex process essential for ciliogenesis, involves the progressive addition of specific proteins to centrioles. CHIBBY (CBY) is a coiled-coil domain protein first described as interacting with β-catenin and involved in Wg-Int (WNT) signaling. We found that, in Drosophila melanogaster, CBY was exclusively expressed in cells that require functional basal bodies, i.e., sensory neurons and male germ cells. CBY was associated with the basal body transition zone (TZ) in these two cell types. Inactivation of cby led to defects in sensory transduction and in spermatogenesis. Loss of CBY resulted in altered ciliary trafficking into neuronal cilia, irregular deposition of proteins on spermatocyte basal bodies, and, consequently, distorted axonemal assembly. Importantly, cby1/1 flies did not show Wingless signaling defects. Hence, CBY is essential for normal basal body structure and function in Drosophila, potentially through effects on the TZ. The function of CBY in WNT signaling in vertebrates has either been acquired during vertebrate evolution or lost in Drosophila.

Cilia and flagella have essential functions in a wide range of organisms. Cilia assembly is dynamic during development and different types of cilia are found in multicellular organisms. How this dynamic and specific assembly is regulated remains an important question in cilia biology. In metazoans, the regulation of the overall expression level of key components necessary for cilia assembly or function is an important way to achieve ciliogenesis control. The FOXJ1 (forkhead box J1) and RFX (regulatory factor X) family of transcription factors have been shown to be important players in controlling ciliary gene expression. They fulfill a complementary and synergistic function by regulating specific and common target genes. FOXJ1 is essential to allow for the assembly of motile cilia in vertebrates through the regulation of genes specific to motile cilia or necessary for basal body apical transport, whereas RFX proteins are necessary to assemble both primary and motile cilia in metazoans, in particular, by regulating genes involved in intraflagellar transport. Recently, different transcription factors playing specific roles in cilia biogenesis and physiology have also been discovered. All these factors are subject to complex regulation to allow for the dynamic and specific regulation of ciliogenesis in metazoans.

Background The weevils Sitophilus spp. are among the most important cosmopolitan pests of stored cereal grains. However, their biology and physiology are poorly understood, mainly because the insect developmental stages take place within cereal grains and because of the lack of gene specific molecular manipulation. Results To gain access to the different insect developmental stages, weevil females were allowed to lay their eggs on starch pellets and hatched embryos were collected by dissolving starch with water. Embryos were transferred between two Glass Plates filled with packed Flour (GPF) to mimic compact texture of the cereal grain, and this system allowed us to recover specific developmental stages. To knockdown the gene expressed in the bacteria-bearing organ (the bacteriome), whole larvae were injected with dsRNA to target the wpgrp1 gene and they were then left to develop for a further 4 days period. Quantitative RT-PCR and Western blot analyses on the bacteriome of these animals revealed a down-regulation of the wpgrp1 expression, both at transcript and protein levels. Conclusion These results demonstrate that whole larval injection with dsRNA results in a high and systemic decrease of both mRNA and protein in the bacteriome tissue. This, along with the possibility of access to the insect developmental stages, opens up a new research avenue for exploring gene specific functions in the cereal weevils.

Background: Drosophila embryogenesis is initiated by 13 rapid syncytial mitotic divisions that do not require zygotic gene activity. This maternally directed cleavage phase of development terminates at the midblastula transition (MBT), at which point the cell cycle slows dramatically, membranes surround the cortical nuclei to form a cellular blastoderm, and zygotic gene expression is first required. Results: We show that embryos lacking Mei-41, a Drosophila homologue of the ATM tumor suppressor, proceed through unusually short syncytial mitoses, fail to terminate syncytial division following mitosis 13, and degenerate without forming cells. A similar cleavage-stage arrest is produced by mutations in grapes, which encodes a homologue of the Checkpoint-1 kinase. We present biochemical, cytological and genetic data indicating that Mei-41 and Grapes are components of a conserved DNA-replication/damage checkpoint pathway that triggers inhibitory phosphorylation of the Cdc2 kinase and mediates resistance to replication inhibitors and DNA-damaging agents. This pathway is nonessential during postembryonic development, but it is required to terminate the cleavage stage at the MBT. Cyclins are required for Cdc2 kinase activity, and mutations in cyclin A and cyclin B bypass the requirement for mei-41 at the MBT. These mutations do not restore wild-type syncytial cell-cycle timing or the embryonic replication checkpoint, however, suggesting that Mei-41-mediated inhibition of Cdc2 has an additional essential function at the MBT. Conclusions: The Drosophila DNA-replication/damage checkpoint pathway can be activated by externally triggered DNA damage or replication defects throughout the life cycle, and under laboratory conditions this inducible function is nonessential. During early embryogenesis, however, this pathway is activated by developmental cues and is required for the transition from maternal to zygotic control of development at the MBT.

In the I-R system of hybrid dysgenesis in Drosophila melanogaster, the transposition frequency of I factor, a LINE element-like retrotransposon, is regulated by the reactivity level of the R mother. This reactivity is a cellular state maternally inherited but chromosomally determined, which has been shown to undergo heritable, cumulative and reversible changes with aging and some environmental conditions. We propose the hypothesis that this reactivity level is one manifestation of an inducible repair-recombination system whose biological role might be analogous to the SOS response in bacteria. In this paper, we show that inhibitors of DNA synthesis and gamma rays enhance the reactivity level in a very similar way. This enhancement is heritable, cumulative and reversible.

In a previous paper, we reported that the reactivity level, which regulates the frequency of transposition of I factor, a LINE element-like retrotransposon, is enhanced by the same agents that induce the SOS response in Escherichia coli. In this report, we describe experimental evidence that, for identical genotypes, the reactivity levels correlate with the sensitivity of oogenesis to gamma rays, measured by the number of eggs laid and by frequency of dominant lethals. This strongly supports the hypothesis that the reactivity level is one manifestation of an inducible DNA repair system taking place in the female germ line of Drosophila melanogaster. The implications of this finding for the understanding of the regulation of I factor are discussed and some other possible biological roles of this system are outlined.

An RFX-binding site is shown to be conserved in the promoters of a subset of ciliary genes and a subsequent screen for this site in two Drosophila species identified novel RFX target genes that are involved in sensory ciliogenesis., Background Regulatory factor X (RFX) transcription factors play a key role in ciliary assembly in nematode, Drosophila and mouse. Using the tremendous advantages of comparative genomics in closely related species, we identified novel genes regulated by dRFX in Drosophila. Results We first demonstrate that a subset of known ciliary genes in Caenorhabditis elegans and Drosophila are regulated by dRFX and have a conserved RFX binding site (X-box) in their promoters in two highly divergent Drosophila species. We then designed an X-box consensus sequence and carried out a genome wide computer screen to identify novel genes under RFX control. We found 412 genes that share a conserved X-box upstream of the ATG in both species, with 83 genes presenting a more restricted consensus. We analyzed 25 of these 83 genes, 16 of which are indeed RFX target genes. Two of them have never been described as involved in ciliogenesis. In addition, reporter construct expression analysis revealed that three of the identified genes encode proteins specifically localized in ciliated endings of Drosophila sensory neurons. Conclusion Our X-box search strategy led to the identification of novel RFX target genes in Drosophila that are involved in sensory ciliogenesis. We also established a highly valuable Drosophila cilia and basal body dataset. These results demonstrate the accuracy of the X-box screen and will be useful for the identification of candidate genes for human ciliopathies, as several human homologs of RFX target genes are known to be involved in diseases, such as Bardet-Biedl syndrome.

The segregation of homologous chromosomes from one another is the essence of meiosis. In many organisms, accurate segregation is ensured by the formation of chiasmata resulting from crossing over. Drosophila melanogaster females use this type of recombination-based system, but they also have mechanisms for segregating achiasmate chromosomes with high fidelity. We describe a P-element mutagenesis and screen in a sensitized genetic background to detect mutations that impair meiotic chromosome pairing, recombination, or segregation. Our screen identified two new recombination-deficient mutations: mei-P22, which fully eliminates meiotic recombination, and mei-P26, which decreases meiotic exchange by 70% in a polar fashion. We also recovered an unusual allele of the ncd gene, whose wild-type product is required for proper structure and function of the meiotic spindle. However, the screen yielded primarily mutants specifically defective in the segregation of achiasmate chromosomes. Although most of these are alleles of previously undescribed genes, five were in the known genes αTubulin67C, CycE, push, and Trl. The five mutations in known genes produce novel phenotypes for those genes.