Morphogen gradient formation and vesicular trafficking

Morphogens are secreted signaling molecules which form spatial concentration gradients while moving away from a restricted source of production. A simple model of gradient formation postulates that the morphogens dilute as they diffuse between cells. In this review we discuss recent data supporting the idea that movement of the morphogen could also occur via vesicular trafficking through the cells. We explore the implications of these results for the control of gradient formation and the determination of the gradient slope which ultimately encodes the coordinates of positional information.     

Two cytochrome P450s in Caenorhabditis elegans are essential for the organization of eggshell,correct execution of meiosis and the polarization of embryo

The role of lipids in the process of embryonic development of Caenorhabditis elegans is still poorly understood. Cytochrome P450s, a class of lipid-modifying enzymes, are good candidates to be involved in the production or degradation of lipids essential for development. We investigated two highly similar cytochrome P450s in C. elegans, cyp-31A2 and cyp-31A3, that are homologs of the gene responsible for Bietti crystalline corneoretinal dystrophy in humans. Depletion of both cytochromes either by RNAi or using a double deletion mutant, led to the failure of establishing the correct polarity of the embryo and to complete the extrusion of the polar bodies during meiosis. In addition, the egg became osmotic sensitive and permeable to dyes. The phenotype of cyp-31A2 or cyp-31A3 is very similar to a class of mutants that have polarization and osmotic defects (POD), thus the genes were renamed to pod-7 and pod-8, respectively. Electron microscopic analysis demonstrated that the activity of pod-7/pod-8 is crucial for the proper assembly of the eggshell and, in particular, for the production of its lipid-rich layer. Using a complementation with lipid extracts, we show that POD-7/POD-8 function together with a NADPH cytochrome P450 reductase, coded by emb-8, and are involved in the production of lipid(s) required for eggshell formation.     

Requirement of sterols in the life cycle of the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans represents an excellent model for studying many aspects of sterol function on the level of a whole organism. Recent studies show that especially two processes in the life cycle of the worm, dauer larva formation and molting, depend on sterols. In both cases, cholesterol or its derivatives seem to act as hormones rather than being structural components of the membrane. Investigations on C. elegans could provide information on the etiology of human diseases that display defects in the transport or metabolism of sterols.     

Larvae of related Diptera species from thermally contrasting habitats exhibit continuous up-regulation of heat shock proteins and high thermotolerance

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name ‘soldier flies’, occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 °C, have constitutively high concentrations of the normally inducible heat-shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi-aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat-shock treatment of S. japonica larvae activates heat-shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.     

LET-767 is required for the production of branched chain and long chain fatty acids in Caenorhabditis elegans

LET-767 from Caenorhabditis elegans belongs to a family of short chain dehydrogenases/reductases and is homologous to 17beta-hydroxysterol dehydrogenases of type 3 and 3-ketoacyl-CoA reductases. Worms subjected to RNA interference (RNAi) of let-767 displayed multiple growth and developmental defects in the first generation and arrested in the second generation as L1 larvae. To determine the function of LET-767 in vivo, we exploited a biochemical complementation approach, in which let-767 (RNAi)-arrested larvae were rescued by feeding with compounds isolated from wild type worms. The arrest was only rescued by the addition of triacylglycerides extracted from worms but not from various natural sources, such as animal fats and plant oils. The mass spectrometric analyses showed alterations in the fatty acid content of triacylglycerides. Essential for the rescue were odd-numbered fatty acids with monomethyl branched chains. The rescue was improved when worms were additionally supplemented with long chain even-numbered fatty acids. Remarkably, let-767 completely rescued the yeast 3-ketoacyl-CoA reductase mutant (ybr159Delta). Because worm ceramides exclusively contain a monomethyl branched chain sphingoid base, we also investigated ceramides in let-767 (RNAi). Indeed, the amount of ceramides was greatly reduced, and unusual sphingoid bases were observed. Taken together, we conclude that LET-767 is a major 3-ketoacyl-CoA reductase in C. elegans required for the bulk production of monomethyl branched and long chain fatty acids, and the developmental arrest in let-767 (RNAi) worms is caused by the deficiency of the former.     

Molecular phylogeny of fungus gnats (Diptera: Mycetophilidae) revisited: position of Manotinae,Metanepsiini, and other enigmatic taxa as inferred from multigene analysis

The phylogeny of selected genera from four subfamilies of fungus gnats (Diptera: Mycetophilidae) – Manotinae, Leiinae, Sciophilinae and Gnoristinae (including Metanepsiini) – is reconstructed based on the combined analysis of five mitochondrial (12S, 16S, COI, COII, cytB) and two nuclear (28S, ITS2) gene markers. Results of the different analyses all support Manotinae as a monophyletic group, with Leiinae as the sister group. Allactoneura DeMeijere is nested in the monophyletic and strongly supported clade of Leiinae. The tribe Metanepsiini is revealed as paraphyletic and the genera Metanepsia Edwards and Chalastonepsia Søli do not appear to be closely related. The genera Docosia Winnertz, Ectrepesthoneura Enderlein, Novakia Strobl and Syntemna Winnertz were placed with a group of genera included traditionally in the Gnoristinae. The monophyly of Dziedzickia Johannsen and Phthinia Winnertz is not supported. The genera of Sciophilinae (excluding Paratinia Mik but including Eudicrana Loew) form a monophyletic group in the Bayesian model.     

A genomic walking method for screening sequence length polymorphism

We adapted a recently developed nonrestrictional, nonligational genome walking method, Universal Fast Walking (UFW), for detection of length polymorphism in the proximal promoter region of genes. We demonstrate its efficacy at discovering naturally occurring transposition into heat‐shock genes of wild Drosophila and show that it surmounts limitations of simple polymerase chain reaction (PCR) approaches. We further present modifications to the standard UFW protocol and provide some guidelines to improve specificity. Although the resultant banding pattern of a standard UFW can be regarded as a DNA fingerprint, many amplicons result from false priming and not real polymorphisms. We describe ways to distinguish between UFW amplicons and false priming products in a high‐throughput assay.