The Homeotic Gene Sex Combs Reduced of Drosophila Melanogaster Is Differentially Regulated in the Embryonic and Imaginal Stages of Development

The Sex combs reduced (Scr) locus is unique among the genes contained within the Antennapedia complex (ANT-C) of Drosophila melanogaster in that it directs functions that are required for both cephalic and thoracic development in the embryo and the adult. Antibodies raised against protein encoded by Scr were used to follow the distribution of this gene product in embryos and imaginal discs of third instar larvae. Analysis of Scr protein accumulation in embryos hemizygous for breakpoint lesions mapping throughout the locus has allowed us to determine that sequences required for establishment of the Scr embryonic pattern are contained within a region of DNA that overlaps with the identified upstream regulatory region of the segmentation gene fushi tarazu (ftz). Gain-of-function mutations in Scr result in the presence of ectopic sex comb teeth on the first tarsal segment of mesothoracic and metathoracic legs of adult males. Heterozygous combinations of gain-of-function alleles with a wild-type Scr gene exhibit no evidence of ectopic protein localization in the second and third thoracic segments of embryos. However, mesothoracic and metathoracic leg imaginal discs can be shown to accumulate ectopically expressed Scr protein, implying a differential regulation of the Scr gene during these two periods of development. Additionally, we have found that the spatial pattern of Scr gene expression in imaginal tissues involved in the development of the adult thorax is governed in part by synapsis of homologous chromosomes in this region of the ANT-C. However, those imaginal discs that arise anteriorly to the prothorax do not appear to be sensitive to this form of gene regulation. Finally, we have demonstrated that the extent of Scr expression is influenced by mutations at the Polycomb (Pc) locus but not by mutant alleles of the zeste (z) gene. Taken together, our data suggests that Scr gene expression is differentially regulated both temporally and spatially in a manner that is sensitive to the structure of the locus.     

Crossover Localisation Is Regulated by the Neddylation Posttranslational Regulatory Pathway

Crossovers (COs) are at the origin of genetic variability, occurring across successive generations, and they are also essential for the correct segregation of chromosomes during meiosis. Their number and position are precisely controlled, however the mechanisms underlying these controls are poorly understood. Neddylation/rubylation is a regulatory pathway of posttranslational protein modification that is required for numerous cellular processes in eukaryotes, but has not yet been linked to homologous recombination. In a screen for meiotic recombination-defective mutants, we identified several axr1 alleles, disrupting the gene encoding the E1 enzyme of the neddylation complex in Arabidopsis. Using genetic and cytological approaches we found that axr1 mutants are characterised by a shortage in bivalent formation correlated with strong synapsis defects. We determined that the bivalent shortage in axr1 is not due to a general decrease in CO formation but rather due to a mislocalisation of class I COs. In axr1, as in wild type, COs are still under the control of the ZMM group of proteins. However, in contrast to wild type, they tend to cluster together and no longer follow the obligatory CO rule. Lastly, we showed that this deregulation of CO localisation is likely to be mediated by the activity of a cullin 4 RING ligase, known to be involved in DNA damage sensing during somatic DNA repair and mouse spermatogenesis. In conclusion, we provide evidence that the neddylation/rubylation pathway of protein modification is a key regulator of meiotic recombination. We propose that rather than regulating the number of recombination events, this pathway regulates their localisation, through the activation of cullin 4 RING ligase complexes. Possible targets for these ligases are discussed.     

The Endo-siRNA Pathway Is Essential for Robust Development of the Drosophila Embryo

Background

Robustness to natural temperature fluctuations is critical to proper development in embryos and to cellular functions in adult organisms. However, mechanisms and pathways which govern temperature compensation remain largely unknown beyond circadian rhythms. Pathways which ensure robustness against temperature fluctuations may appear to be nonessential under favorable, uniform environmental conditions used in conventional laboratory experiments where there is little variation for which to compensate. The endo-siRNA pathway, which produces small double-stranded RNAs in Drosophila, appears to be nonessential for robust development of the embryo under ambient uniform temperature and to be necessary only for viral defense. Embryos lacking a functional endo-siRNA pathway develop into phenotypically normal adults. However, we hypothesized that small RNAs may regulate the embryo''s response to temperature, as a ribonucleoprotein complex has been previously shown to mediate mammalian cell response to heat shock.

Principal Findings

Here, we show that the genes DICER-2 and ARGONAUTE2, which code for integral protein components of the endo-siRNA pathway, are essential for robust development and temperature compensation in the Drosophila embryo when exposed to temperature perturbations. The regulatory functions of DICER-2 and ARGONAUTE2 were uncovered by using microfluidics to expose developing Drosophila embryos to a temperature step, in which each half of the embryo develops at a different temperature through developmental cycle 14. Under this temperature perturbation, dicer-2 or argonaute2 embryos displayed abnormal segmentation. The abnormalities in segmentation are presumably due to the inability of the embryo to compensate for temperature-induced differences in rate of development and to coordinate developmental timing in the anterior and posterior halves. A deregulation of the length of nuclear division cycles 10–14 is also observed in dicer-2 embryos at high temperatures.

Conclusions

Results presented herein uncover a novel function of the endo-siRNA pathway in temperature compensation and cell cycle regulation, and we hypothesize that the endo-siRNA pathway may regulate the degradation of maternal cell cycle regulators. Endo-siRNAs may have a more general role buffering against environmental perturbations in other organisms.     

Neuronal Development in Caenorhabditis elegans Is Regulated by Inhibition of an MLK MAP Kinase Pathway

We show that loss-of-function mutations in kinases of the MLK-1 pathway (mlk-1, mek-1, and kgb-1/jnk) function cell-autonomously in neurons to suppress defects in synapse formation and axon termination caused by rpm-1 loss of function. Our genetic analysis also suggests that the phosphatase PPM-1, like RPM-1, is a potential inhibitor of kinases in the MLK-1 pathway.     

DNA-Damage-Induced Nuclear Export of Precursor MicroRNAs Is Regulated by the ATM-AKT Pathway

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Abscission Is Regulated by the ESCRT-III Protein Shrub in Drosophila Germline Stem Cells

Abscission is the final event of cytokinesis that leads to the physical separation of the two daughter cells. Recent technical advances have allowed a better understanding of the cellular and molecular events leading to abscission in isolated yeast or mammalian cells. However, how abscission is regulated in different cell types or in a developing organism remains poorly understood. Here, we characterized the function of the ESCRT-III protein Shrub during cytokinesis in germ cells undergoing a series of complete and incomplete divisions. We found that Shrub is required for complete abscission, and that levels of Shrub are critical for proper timing of abscission. Loss or gain of Shrub delays abscission in germline stem cells (GSCs), and leads to the formation of stem-cysts, where daughter cells share the same cytoplasm as the mother stem cell and cannot differentiate. In addition, our results indicate a negative regulation of Shrub by the Aurora B kinase during GSC abscission. Finally, we found that Lethal giant discs (lgd), known to be required for Shrub function in the endosomal pathway, also regulates the duration of abscission in GSCs.     

Timing of Expression of a Gene in the Adult Drosophila Is Regulated by Mechanisms Independent of Temperature and Metabolic Rate

The examination of β-galactosidase (β-gal) expression in the third segment of the antenna of the 2216 enhancer trap line in Drosophila melanogaster reveals two distinct spatial and temporal regulatory patterns of expression during adult life. Type I expression is characterized by a decline in the level of β-gal expression with increasing age. Starting from a maximal level of expression at the time of adult emergence, there is a decrease in the number of cells that express β-gal so that by 40-50 days of adult life few cells express β-gal. Varying the ambient temperature and using hyperactivity mutants (Hyperkinetic(1), Shaker(5)) demonstrates that the rate of this decline is independent of temperature and metabolic rate. Type II expression is distinctly different in spatial distribution and temporal regulation from the first pattern. Type II expression is restricted in the antenna to a small (<20-30) set of cells whose level of expression changes in a periodic manner with time. The regulation of this periodicity appears to be linked to ambient temperature.     

The Imprinted Gene Neuronatin Is Regulated by Metabolic Status and Associated With Obesity

Using restriction fragment differential display (RFDD) technology, we have identified the imprinted gene neuronatin (Nnat) as a hypothalamic target under the influence of leptin. Nnat mRNA expression is decreased in several key appetite regulatory hypothalamic nuclei in rodents with impaired leptin signaling and during fasting conditions. Furthermore, peripheral administration of leptin to ob/ob mice normalizes hypothalamic Nnat expression. Comparative immunohistochemical analysis of human and rat hypothalami demonstrates that NNAT protein is present in anatomically equivalent nuclei, suggesting human physiological relevance of the gene product(s). A putative role of Nnat in human energy homeostasis is further emphasized by a consistent association between single nucleotide polymorphisms (SNPs) in the human Nnat gene and severe childhood and adult obesity.     

The Tolkin Gene Is a Tolloid/Bmp-1 Homologue That Is Essential for Drosophila Development

The Drosophila decapentaplegic (dpp) gene, a member of the tranforming growth factor β superfamily of growth factors, is critical for specification of the embryonic dorsal-ventral axis, for proper formation of the midgut, and for formation of Drosophila adult structures. The Drosophila tolloid gene has been shown to genetically interact with dpp. The genetic interaction between tolloid and dpp suggests a model in which the tolloid protein participates in a complex containing the DPP ligand, its protease serving to activate DPP, either directly or indirectly. We report here the identification and cloning of another Drosophila member of the tolloid/bone morphogenic protein (BMP) 1 family, tolkin, which is located 700 bp 5' to tolloid. Its overall structure is like tolloid, with an N-terminal metalloprotease domain, five complement subcomponents C1r/C1s, Uegf, and Bmp1 (CUB) repeats and two epidermal growth factor (EGF) repeats. Its expression pattern overlaps that of tolloid and dpp in early embryos and diverges in later stages. In larval tissues, both tolloid and tolkin are expressed uniformly in the imaginal disks. In the brain, both tolloid and tolkin are expressed in the outer proliferation center, whereas tolkin has another stripe of expression near the outer proliferation center. Analysis of lethal mutations in tolkin indicate it is vital during larval and pupal stages. Analysis of its mutant phenotypes and expression patterns suggests that its functions may be mostly independent of tolloid and dpp.     

DNA Repair Pathway Choice Is Influenced by the Health of Drosophila melanogaster

In nature, individuals vary tremendously in condition and this may be an important source of variation in mutation rate. Condition is likely to affect cell state and thereby impact the amount of DNA damage sustained and/or the way it is repaired. Here, we focus on DNA repair. If low-condition individuals are less capable of devoting the same level of resources to accurate repair, they may suffer higher mutation rates. However, repair decisions are also governed by various aspects of cell physiology, which may render the prediction that “higher-condition individuals use better repair mechanisms” too simplistic. We use a larval diet manipulation in Drosophila melanogaster to create high- and low-condition individuals and then contrast their relative usage of three repair pathways [homologous recombination (HR), single-strand annealing (SSA), and nonhomologous end joining (NHEJ)] that differ in their mechanistic requirements and their mutational consequences. We find that low-condition flies are more likely than high-condition flies to use the most conservative of these three repair pathways, suggesting that physiological constraints on repair pathway usage may be more important than energetic costs. We also show that the repair differences between high- and low-condition flies resemble those between young and old flies, suggesting the underlying mechanisms may be similar. Finally, we observe that the effect of larval diet on adult repair increases as flies age, indicating that developmental differences early in life can have long-lasting consequences.     

PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix

Loss-of-function mutations in PINK1, which encodes a mitochondrially targeted serine/threonine kinase, result in an early-onset heritable form of Parkinson''s disease. Previous work has shown that PINK1 is constitutively degraded in healthy cells, but selectively accumulates on the surface of depolarized mitochondria, thereby initiating their autophagic degradation. Although PINK1 is known to be a cleavage target of several mitochondrial proteases, whether these proteases account for the constitutive degradation of PINK1 in healthy mitochondria remains unclear. To explore the mechanism by which PINK1 is degraded, we performed a screen for mitochondrial proteases that influence PINK1 abundance in the fruit fly Drosophila melanogaster. We found that genetic perturbations targeting the matrix-localized protease Lon caused dramatic accumulation of processed PINK1 species in several mitochondrial compartments, including the matrix. Knockdown of Lon did not decrease mitochondrial membrane potential or trigger activation of the mitochondrial unfolded protein stress response (UPR^mt), indicating that PINK1 accumulation in Lon-deficient animals is not a secondary consequence of mitochondrial depolarization or the UPR^mt. Moreover, the influence of Lon on PINK1 abundance was highly specific, as Lon inactivation had little or no effect on the abundance of other mitochondrial proteins. Further studies indicated that the processed forms of PINK1 that accumulate upon Lon inactivation are capable of activating the PINK1-Parkin pathway in vivo. Our findings thus suggest that Lon plays an essential role in regulating the PINK1-Parkin pathway by promoting the degradation of PINK1 in the matrix of healthy mitochondria.     

Drosophila Cyclin G Is a Regulator of the Notch Signalling Pathway during Wing Development

Notch signalling regulates a multitude of differentiation processes during Drosophila development. For example, Notch activity is required for proper wing vein differentiation which is hampered in mutants of either the receptor Notch, the ligand Delta or the antagonist Hairless. Moreover, the Notch pathway is involved in several aspects of Drosophila oogenesis as well. We have identified Drosophila Cyclin G (CycG) as a molecular interaction partner of Hairless, the major antagonist in the Notch signalling pathway, in vitro and in vivo. Loss of CycG was shown before to cause female sterility and to disturb the architecture of the egg shell. Nevertheless, Notch dependent processes during oogenesis appeared largely unaffected in cycG mutant egg chambers. Loss of CycG modified the dominant wing phenotypes of Notch, Delta and Hairless mutants. Whereas the Notch loss of function phenotype was ameliorated by a loss of CycG, the phenotypes of either Notch gain of function or of Delta or Hairless loss of function were enhanced. In contrast, loss of CycG had only a minor effect on the wing vein phenotype of mutants affecting the EGFR signalling pathway emphasizing the specificity of the interaction of CycG and Notch pathway members.