Prooxidative chain transfer activity by thiol groups in biological systems.

Cysteine is arguably the best-studied biological amino acid, whose thiol group frequently participates in catalysis or ligand binding by proteins. Still, cysteine's unusual biological distribution has remained mysterious, being strikingly underrepresented in transmembrane domains and on accessible protein surfaces, particularly in aerobic life forms ("cysteine anomaly"). Noting that lipophilic thiols have been used for decades as radical chain transfer agents in polymer chemistry, we speculated that the rapid formation of thiyl radicals in hydrophobic phases might provide a rationale for the cysteine anomaly. Hence, we have investigated the effects of dodecylthiol and related compounds in isolated biomembranes, cultivated human cells and whole animals (C. elegans). We have found that lipophilic thiols at micromolar concentrations were efficient accelerators, but not inducers of lipid peroxidation, catalyzed fatty acid isomerization to trans-fatty acids, and evoked a massive cellular stress response related to protein and DNA damage. These effects were specific for lipophilic thiols and were absent with thioethers, alcohols or hydrophilic compounds. Catalytic chain transfer activity by thiyl radicals appears to have deeply influenced the structural biology of life as reflected in the cysteine anomaly. Chain transfer agents represent a novel class of biological cytotoxins that selectively accelerate oxidative damage in vivo.

The transcription factor Zfh1 is involved in the regulation of neuropeptide expression and growth of larval neuromuscular junctions in Drosophila melanogaster.

Different aspects of neural development are tightly regulated and the underlying mechanisms have to be transcriptionally well controlled. Here we present evidence that the transcription factor Zfh1, the Drosophila member of the conserved zfh1 gene family, is important for different steps of neuronal differentiation. First, we show that late larval expression of the neuropeptide FMRFamide is dependent on correct levels of Zfh1 and that this regulation is presumably direct via a conserved zfh1 homeodomain binding site in the FMRFamide enhancer. Using MARCM analysis we additionally examined the requirement for Zfh1 during embryonic and larval stages of motoneuron development. We could show that Zfh1 cell autonomously regulates motoneuronal outgrowth and larval growth of neuromuscular junctions (NMJs). In addition, we find that the growth of NMJs is dependent on the dosage of Zfh1, suggesting it to be a downstream effector of the known NMJ size regulating pathways.

Connecting temporal identity to mitosis: the regulation of Hunchback in Drosophila neuroblast lineages.

Both in vertebrates and invertebrates, neural stem cells generate different cell types at different times during development. It has been suggested that this process depends on temporal identity transitions of neural progenitors, but the underlying mechanism has not been resolved, yet. Recently, Drosophila neuroblasts (NBs) have been shown to be an excellent model system to investigate this subject. Here, changes in temporal identity are regulated by sequential and transient expression of transcription factors in the NB, such as Hunchback (Hb) and Krüppel (Kr). The temporal expression profile is maintained in the progeny. Hb is expressed first and thus defines the earliest identity in a given lineage. Transition to Kr requires the termination of hb expression, which occurs in response to mitosis, and is mediated by Seven-up (Svp). Recent results provided evidence that the dependency of Svp activity on mitosis could be due to an inhibition of the nuclear export of svp mRNA. Furthermore, the maintenance of hb expression in the GMC is regulated by the activity of Prospero (Pros), a transcription factor which asymmetrically segregates into the GMC during mitosis and inhibits Svp activity on both, the transcriptional and posttranscriptional level. These results give first insights as to how temporal cell fate specification can be correlated with mitosis.

Timing of identity: spatiotemporal regulation of hunchback in neuroblast lineages of Drosophila by Seven-up and Prospero.

Neural stem cells often generate different cell types in a fixed birth order as a result of temporal specification of the progenitors. In Drosophila, the first temporal identity of most neural stem cells (neuroblasts) in the embryonic ventral nerve cord is specified by the transient expression of the transcription factor Hunchback. When reaching the next temporal identity, this expression is switched off in the neuroblasts by seven up (svp) in a mitosis-dependent manner, but is maintained in their progeny (ganglion mother cells). We show that svp mRNA is already expressed in the neuroblasts before this division. After mitosis, Svp protein accumulates in both cells, but the downregulation of hunchback (hb) occurs only in the neuroblast. In the ganglion mother cell, svp is repressed by Prospero, a transcription factor asymmetrically localised to this cell during mitosis. Thus, the differential regulation of hb between the neuroblasts and the ganglion mother cells is achieved by a mechanism that integrates information created by the asymmetric distribution of a cell-fate determinant upon mitosis (Prospero) and a transcriptional repressor present in both cells (Seven-up). Strikingly, although the complete downregulation of hb is mitosis dependent, the lineage-specific timing of svp upregulation is not.

Retrieval of vertical constituents and temperature profiles from passive submillimeter wave limb observations of the Martian atmosphere: a feasibility study.

The investigation of the Martian atmosphere is of key importance for an understanding of the planets present and past. Passive limb observations of thermal radiation at submillimeter wavelengths in the 320-350-GHz range by use of a state-of-the-art satellite receiver on a low Mars orbit allow important parameters such as the mixing ratios of H2O, HDO, 12CO, 13CO, O3, and H2O2 as well as the thermal profile to be retrieved with high precision and unprecedented vertical range and resolution, providing valuable information for better understanding of the planet's water cycle, atmospheric dynamics, and photochemistry. The feasibility of these kinds of measurement is demonstrated by means of model simulations based on realistic atmospheric, spectroscopic, and instrumental parameters. Temperature can be retrieved to approximately 90 km with half-scale height vertical resolution from single-scan measurements of emission lines of the long-lived species 12CO and 13CO. The global water-vapor distribution can be measured even under dry or wet conditions with good vertical resolution from the surface to approximately 45 km, and simultaneous observations of HDO allow useful information on the D/H ratio up to an altitude of approximately 30 km to be derived. The sensitivity of the limb-sounding technique also permits information on the photochemically important minor species O3, and H2O2 to be obtained. It is shown that spectral averaging may improve precision, altitude range, and resolution of the retrieved profiles. Other frequency bands are explored, and the 435-465-GHz range is suggested as a possible alternative to the 320-350-GHz range.

Hunchback is required for the specification of the early sublineage of neuroblast 7-3 in the Drosophila central nervous system.

The Drosophila ventral nerve cord (VNC) derives from neuroblasts (NBs), which mostly divide in a stem cell mode and give rise to defined NB lineages characterized by specific sets of sequentially generated neurons and/or glia cells. To understand how different cell types are generated within a NB lineage, we have focused on the NB7-3 lineage as a model system. This NB gives rise to four individually identifiable neurons and we show that these cells are generated from three different ganglion mother cells (GMCs). The finding that the transcription factor Hunchback (Hb) is expressed in the early sublineage of NB7-3, which consists of the early NB and the first GMC (GMC7-3a) and its progeny (EW1 and GW), prompted us to investigate its possible role in NB7-3 lineage development. Our analysis revealed that loss of hb results in a lack of the normally Hb-positive neurons, while the later-born neurons (designated as EW2 and EW3) are still present. However, overexpression of hb in the whole lineage leads to additional cells with the characteristics of GMC7-3a-derived neurons, at the cost of EW2 and EW3. Thus, hb is an important determinant in specifying early sublineage identity in the NB7-3 lineage. Using Even-skipped (Eve) as a marker, we have additionally shown that hb is also needed for the determination and/or differentiation of several other early-born neurons, indicating that this gene is an important player in sequential cell fate specification within the Drosophila CNS.

Distribution, classification, and development ofDrosophila glial cells in the late embryonic and early larval ventral nerve cord.

To facilitate the investigation of glial development inDrosophila, we present a detailed description of theDrosophila glial cells in the ventral nerve cord. A GAL4 enhancer-trap screen for glial-specific expression was performed. Using UAS-lacZ and UAS-kinesin-lacZ as reporter constructs, we describe the distribution and morphology of the identified glial cells in the fully differentiated ventral nerve cord of first-instar larvae just after hatching. The three-dimensional structure of the glial network was reconstructed using a computer. Using the strains with consistent GAL4 expression during late embryogenesis, we traced back the development of the identified cells to provide a glial map at embryonic stage 16. We identify typically 60 (54-64) glial cells per abdominal neuromere both in embryos and early larvae. They are divided into six subtypes under three categories: surface-associated glia (16-18 subperineurial glial cells and 6-8 channel glial cells), cortex-associated glia (6-8 cell body glial cells), and neuropile-associated glia (8-10 nerve root glial cells, 14-16 interface glial cells, and 3-4 midline glial cells). The proposed glial classification system is discussed in comparison with previous insect glial classifications.

Charting the Drosophila neuropile: a strategy for the standardised characterisation of genetically amenable neurites.

Insect neurons are individually identifiable and have been used successfully to study principles of the formation and function of neuronal circuits. In the fruitfly Drosophila, studies on identifiable neurons can be combined with efficient genetic approaches. However, to capitalise on this potential for studies of circuit formation in the CNS of Drosophila embryos or larvae, we need to identify pre- and postsynaptic elements of such circuits and describe the neuropilar territories they occupy. Here, we present a strategy for neurite mapping, using a set of evenly distributed landmarks labelled by commercially available anti-Fasciclin2 antibodies which remain comparatively constant between specimens and over developmental time. By applying this procedure to neurites labelled by three Gal4 lines, we show that neuritic territories are established in the embryo and maintained throughout larval life, although the complexity of neuritic arborisations increases during this period. Using additional immunostainings or dye fills, we can assign Gal4-targeted neurites to individual neurons and characterise them further as a reference for future experiments on circuit formation. Using the Fasciclin2-based mapping procedure as a standard (e.g., in a common database) would facilitate studies on the functional architecture of the neuropile and the identification of candiate circuit elements.

Evidence for differential and redundant function of the Sox genes Dichaete and SoxN during CNS development in Drosophila.

Group B Sox-domain proteins encompass a class of conserved DNA-binding proteins expressed from the earliest stages of metazoan CNS development. In all higher organisms studied to date, related Group B Sox proteins are co-expressed in the developing CNS; in vertebrates there are three (Sox1, Sox2 and Sox3) and in Drosophila there are two (SoxNeuro and Dichaete). It has been suggested there may be a degree of functional redundancy in Sox function during CNS development. We describe the CNS phenotype of a null mutation in the Drosophila SoxNeuro gene and provide the first direct evidence for both redundant and differential Sox function during CNS development in Drosophila. In the lateral neuroectoderm, where SoxNeuro is uniquely expressed, SoxNeuro mutants show a loss or reduction of achaete expression as well as a loss of many correctly specified lateral neuroblasts. By contrast, in the medial neuroectoderm, where the expression of SoxNeuro and Dichaete overlaps, the phenotypes of both single mutants are mild. In accordance with an at least partially redundant function in that region, SoxNeuro/Dichaete double mutant embryos show a severe neural hypoplasia throughout the central nervous system, as well as a dramatic loss of achaete expressing proneural clusters and medially derived neuroblasts. However, the finding that Dichaete and SoxN exhibit opposite effects on achaete expression within the intermediate neuroectoderm demonstrates that each protein also has region-specific unique functions during early CNS development in the Drosophila embryo.

The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells.

In Drosophila, neurons and glial cells are produced by neural precursor cells called neuroblasts (NBs), which can be individually identified. Each NB generates a characteristic cell lineage specified by a precise spatiotemporal control of gene expression within the NB and its progeny. Here we show that the homeobox genes ladybird early and ladybird late are expressed in subsets of cells deriving from neuroblasts NB 5-3 and NB 5-6 and are essential for their correct development. Our analysis revealed that ladybird in Drosophila, like their vertebrate orthologous Lbx1 genes, play an important role in cell fate specification processes. Among those cells that express ladybird are NB 5-6-derived glial cells. In ladybird loss-of-function mutants, the NB 5-6-derived exit glial cells are absent while overexpression of these genes leads to supernumerary glial cells of this type. Furthermore, aberrant glial cell positioning and aberrant spacing of axonal fascicles in the nerve roots observed in embryos with altered ladybird function suggest that the ladybird genes might also control directed cell movements and cell-cell interactions within the developing Drosophila ventral nerve cord.

Characterization of Drosophila hemoglobin. Evidence for hemoglobin-mediated respiration in insects.

In contrast to previous assumptions, the fruit fly Drosophila melanogaster possesses hemoglobin. This respiratory protein forms a monomer of about 17 kDa that is not exported into the hemolymph. Recombinant Drosophila hemoglobin displays a typical hexacoordinated deoxy spectrum and binds oxygen with an affinity of 0.12 torr. Four different hemoglobin transcripts have been identified, which are generated by two distinct promoters of the hemoglobin (glob1) gene but are identical in their coding regions. Putative binding sites for hypoxia-regulated transcription factors have been identified in the gene. Hemoglobin synthesis in Drosophila is mainly associated with the tracheal system and the fat body. This suggests that oxygen supply in insects may be more complex than thought previously and may depend on hemoglobin-mediated oxygen transport and storage in addition to simple diffusion.