Revisiting bicoid function: complete inactivation reveals an additional fundamental role in Drosophila egg geometry specification.

INTRODUCTION: The bicoid (bcd) gene in Drosophila has served as a paradigm for a morphogen in textbooks for decades. Discovered in 1986 as a mutation affecting anterior development in the embryo, its expression pattern as a protein gradient later confirmed the prediction from transplantation experiments. These experiments suggested that the protein fulfills the criteria of a true morphogen, with the existence of a homeodomain crucial for activation of genes along the anterior-posterior axis, based on the concentration of the morphogen. The bcd gene undergoes alternative splicing, resulting in, among other isoforms, a small and often neglected isoform with low abundance, which lacks the homeodomain, termed small bicoid (smbcd). Most importantly, all known classical strong bcd alleles used in the past to determine bcd function apparently do not affect the function of this isoform. RESULTS: To overcome the uncertainty regarding which isoform regulates what, I removed the bcd locus entirely using CRISPR technology. bcdCRISPR eggs exhibited a short and round appearance. The phenotype could be ascribed to smbcd because all bcd alleles affecting the function of the major transcript, termed large bicoid (lgbcd) showed normally sized eggs. Several patterning genes for the embryo showed expression in the oocyte, and their expression patterns were altered in bcdCRISPR oocytes. In bcdCRISPR embryos, all downstream segmentation genes showed altered expression patterns, consistent with the expression patterns in "classical" alleles; however, due to the altered egg geometry resulting in fewer blastoderm nuclei, additional constraints came into play, further affecting their expression patterns. CONCLUSIONS: This study unveils a novel and fundamental role of bcd in shaping the egg's geometry. This discovery demands a comprehensive revision of our understanding of this important patterning gene and prompts a reevaluation of past experiments conducted under the assumption that bcd mutants were bcdnull-mutants.

Characterization of Drosophila Nidogen/entactin reveals roles in basement membrane stability, barrier function and nervous system patterning.

Basement membranes (BMs) are specialized layers of extracellular matrix (ECM) mainly composed of Laminin, type IV Collagen, Perlecan and Nidogen/entactin (NDG). Recent in vivo studies challenged the initially proposed role of NDG as a major ECM linker molecule by revealing dispensability for viability and BM formation. Here, we report the characterization of the single Ndg gene in Drosophila. Embryonic Ndg expression was primarily observed in mesodermal tissues and the chordotonal organs, whereas NDG protein localized to all BMs. Although loss of Laminin strongly affected BM localization of NDG, Ndg-null mutants exhibited no overt changes in the distribution of BM components. Although Drosophila Ndg mutants were viable, loss of NDG led to ultrastructural BM defects that compromised barrier function and stability in vivo Moreover, loss of NDG impaired larval crawling behavior and reduced responses to vibrational stimuli. Further morphological analysis revealed accompanying defects in the larval peripheral nervous system, especially in the chordotonal organs and the neuromuscular junction (NMJ). Taken together, our analysis suggests that NDG is not essential for BM assembly but mediates BM stability and ECM-dependent neural plasticity during Drosophila development.

Modulating the bicoid gradient in space and time.

BACKGROUND: The formation of the Bicoid (Bcd) gradient in the early Drosophila is one of the most fascinating observations in biology and serves as a paradigm for gradient formation, yet its mechanism is still not fully understood. Two distinct models were proposed in the past, the SDD and the ARTS model. RESULTS: We define novel cis- and trans-acting factors that are indispensable for gradient formation. The first one is the poly A tail length of the bcd mRNA where we demonstrate that it changes not only in time, but also in space. We show that posterior bcd mRNAs possess a longer poly tail than anterior ones and this elongation is likely mediated by wispy (wisp), a poly A polymerase. Consequently, modulating the activity of Wisp results in changes of the Bcd gradient, in controlling downstream targets such as the gap and pair-rule genes, and also in influencing the cuticular pattern. Attempts to modulate the Bcd gradient by subjecting the egg to an extra nuclear cycle, i.e. a 15th nuclear cycle by means of the maternal haploid (mh) mutation showed no effect, neither on the appearance of the gradient nor on the control of downstream target. This suggests that the segmental anlagen are determined during the first 14 nuclear cycles. Finally, we identify the Cyclin B (CycB) gene as a trans-acting factor that modulates the movement of Bcd such that Bcd movement is allowed to move through the interior of the egg. CONCLUSIONS: Our analysis demonstrates that Bcd gradient formation is far more complex than previously thought requiring a revision of the models of how the gradient is formed.

The centenary of Hereditas - almost a fairytale.

This is the Editorial for a series of articles illuminating the history and success story of Hereditas, one of the oldest journals in the area of genetics. For this reason, we invite you to read this special series on a journal with a distinct reputation and which can celebrate its Centenary in 2020.

bicoid RNA localization requires the trans-Golgi network.

BACKGROUND: The formation of the bicoid (bcd) mRNA gradient is a crucial step for Bcd protein gradient formation in Drosophila. In the past, a microtubule (MT)-based cortical network had been shown to be indispensable for bcd mRNA transport to the posterior. RESULTS: We report the identification of a MT-binding protein CLASP/Chb as the first component associated with this cortical MT network. Since CLASPs in vertebrates were shown to serve as an acentriolar microtubule organization center (aMTOC) in concert with trans-Golgi proteins, we examined the effect of the Drosophila trans-Golgins on bcd localization and gradient formation. Using a genetic approach, we demonstrate that the Drosophila trans-Golgins dGCC88, dGolgin97 and dGCC185 indeed affect bcd mRNA localization during oocyte development. Consequently, the bcd mRNA is already mislocalized before the egg is fertilized. The expression domains of genes downstream of the hierarchy of bcd, e.g. of the gap gene empty spiracles or of the pair-rule gene even-skipped are changed, indicating an altered segmental anlagen, due to a faulty bcd gradient. Thus, at the end of embryogenesis, trans-Golgin mutants show bcd-like cuticle phenotypes. CONCLUSIONS: Our data provides evidence that the Golgi as a cellular member of the secretory pathway exerts control on bcd localization which indicates that bcd gradient formation is probably more intricate than previously presumed.

Seeing is believing: the Bicoid protein reveals its path.

In this commentary, I will review the latest findings on the Bicoid (Bcd) morphogen in Drosophila, a paradigm for gradient formation taught to biology students for more than two decades. "Seeing is believing" also summarizes the erroneous steps that were needed to elucidate the mechanisms of gradient formation and the path of movement of Bcd. Initially proclaimed as a dogma in 1988 and later incorporated into the SDD model where the broad diffusion of Bcd throughout the embryo was the predominant step leading to gradient formation, the SDD model was irrefutable for more than two decades until first doubts were raised in 2007 regarding the diffusion properties of Bcd associated with the SDD model. This led to re-thinking of the issue and the definition of a new model, termed the ARTS model which could explain most of the physical constraints that were inherently associated with the SDD model. In the ARTS model, gradient formation is mediated by the mRNA which is redistributed along cortical microtubules to form a mRNA gradient which is translated to form the protein gradient. Contrary to the SDD model, there is no Bcd diffusion from the tip. The ARTS model is also compatible with the observed cortical movement of Bcd. I will critically compare the SDD and the ARTS models as well as other models, analyze the major differences, and highlight the path where Bcd is localized during early nuclear cycles.

Drosophila exoribonuclease nibbler is a tumor suppressor, acts within the RNAi machinery and is not enriched in the nuage during early oogenesis.

BACKGROUND: micro RNAs (miRNAs) are important regulators of many biological pathways. A plethora of steps are required to form, from a precursor, the mature miRNA that eventually acts on its target RNA to repress its expression or to inhibit translation. Recently, Drosophila nibbler (nbr) has been shown to be an important player in the maturation process of miRNA and piRNA. Nbr is an exoribonuclease which helps to shape the 3' end of miRNAs by trimming the 3' overhang to a final length. RESULTS: In contrast to previous reports on the localization of Nbr, we report that 1) Nbr is expressed only during a short time of oogenesis and appears ubiquitously localized within oocytes, and that 2) Nbr was is not enriched in the nuage where it was shown to be involved in piwi-mediated mechanisms. To date, there is little information available on the function of nbr for cellular and developmental processes. Due to the fact that nbr mutants are viable with minor deleterious effects, we used the GAL4/UAS over-expression system to define novel functions of nbr. We disclose hitherto unknown functions of nbr 1) as a tumor suppressor and 2) as a suppressor of RNAi. Finally, we confirm that nbr is a suppressor of transposon activity. CONCLUSIONS: Our data suggest that nbr exerts much more widespread functions than previously reported from trimming 3' ends of miRNAs only.

Ancient role of ten-m/odz in segmentation and the transition from sequential to syncytial segmentation.

BACKGROUND: Until recently, mechanisms of segmentation established for Drosophila served as a paradigm for arthropod segmentation. However, with the discovery of gene expression waves in vertebrate segmentation, another paradigm based on oscillations linked to axial growth was established. The Notch pathway and hairy delay oscillator are basic components of this mechanism, as is the wnt pathway. With the establishment of oscillations during segmentation of the beetle Tribolium, a common segmentation mechanism may have been present in the last common ancestor of vertebrates and arthropods. However, the Notch pathway is not involved in segmentation of the initial Drosophila embryo. In arthropods, the engrailed, wingless pair has a much more conserved function in segmentation than most of the hierarchy established for Drosophila. RESULTS: Here, we work backwards from this conserved pair by discussing possible mechanisms which could have taken over the role of the Notch pathway. We propose a pivotal role for the large transmembrane protein Ten-m/Odz. Ten-m/Odz may have had an ancient role in cell-cell communication, parallel to the Notch and wnt pathways. The Ten-m protein binds to the membrane with properties which resemble other membrane-based biochemical oscillators. CONCLUSION: We propose that such a simple transition could have formed the initial scaffold, on top of which the hierarchy, observed in the syncytium of dipterans, could have evolved.

The Effect of Enamel Sandblasting on Enhancing Bond Strength of Orthodontic Brackets: A Systematic Review and Meta-analysis.

PURPOSE: To critically appraise the evidence regarding the effect of enamel sandblasting on the bond strength of orthodontic brackets on either the labial or lingual tooth surface. MATERIALS AND METHODS: An electronic database search of published and unpublished literature was performed. Search terms included sandblasting, enamel abrasion, tooth surface, bond strength, bond failure, and adhesive remnant; data were extracted in standardized piloted forms. Risk of bias was assessed using the Cochrane risk of bias tool, adapted for in vitro studies where necessary. RESULTS: Of the 81 articles initially retrieved, 13 were eligible for inclusion in the systematic review. All of the latter were in vitro studies with unclear risk of bias primarily due to unclear reporting of blinding of outcome assessors. Eight studies assessed the combined effect of enamel sandblasting and etching, while only five evaluated the isolated effect of sandblasting on the buccal enamel surface. In view of the apparently heterogeneous study settings, intervention protocols, specimen preparation and storage sequences, only two studies were deemed eligible for quantitative synthesis. Random effects meta-analysis revealed no evidence to support sandblasting prior to etching over etching alone with regard to shear bond strength of orthodontic brackets bonded in vitro to lingual enamel surfaces of extracted premolars (standardized mean difference: 0.36; 95% CI: -0.21, 0.94; p = 0.22). CONCLUSIONS: The findings of the present study cannot support lingual enamel sandblasting prior to etching for augmentation of the bond strength of orthodontic brackets.

Intra-cavity frequency-doubled mode-locked semiconductor disk laser at 325 nm.

We present a passively mode-locked semiconductor disk laser (SDL) emitting at 650nm with intra-cavity second harmonic generation to the ultraviolet (UV) spectral range. Both the gain and the absorber structure contain InP quantum dots (QDs) as active material. In a v-shaped cavity using the semiconductor samples as end mirrors, a beta barium borate (BBO) crystal is placed in front of the semiconductor saturable absorber mirror (SESAM) for pulsed UV laser emission in one of the two outcoupled beams. Autocorrelation (AC) measurements at the fundamental wavelength reveal a FWHM pulse duration of 1.22ps. With a repetition frequency of 836MHz, the average output power is 10mW per beam for the red emission and 0.5mW at 325nm.

Bush encroachment control and risk management in semi-arid rangelands.

We study the role of bush encroachment control for a farmer's income and income risk in a stochastic ecological-economic model of grazing management in semi-arid rangelands. In particular, we study debushing as an instrument of risk management that complements the choice of an adaptive grazing management strategy for that sake. We show that debushing, while being a good practice for increasing the mean pasture productivity and thus expected income, also increases the farmer's income risk. The optimal extent of debushing for a risk-averse farmer is thus determined from balancing the positive and negative consequences of debushing on intertemporal and stochastic farm income.

Effect of intraoral aging on the setting status of resin composite and glass ionomer orthodontic adhesives.

INTRODUCTION: The aim of this study was to assess the effect of intraoral aging on the setting status of a resin composite and a glass ionomer adhesive, relative to control specimens stored in water. METHODS: Metallic brackets were bonded with resin composite orthodontic adhesive (Transbond XT; 3M Unitek, Monrovia, Calif) or a glass ionomer cement (Fuji I; GC, Tokyo, Japan) to recently extracted premolars and kept in water for 6 months. The same materials were also bonded to the premolars of orthodontic patients. After 6 months, the teeth were carefully extracted, with the brackets intact on their buccal surfaces. All teeth were embedded in epoxy resin and sectioned buccolingually. Fourier transform infrared microscopy and Raman microscopy were used for the estimation of the degree of cure in the composite and the salt yield in the glass ionomer adhesives. RESULTS: The control samples of the composite showed significantly lower degrees of cure than did the retrieved specimens (52.40% ± 3.21% vs 57.62% ± 1.32% by Fourier transform infrared microscopy, and 61.40% ± 2.61% vs 67.40% ± 3.44% by Raman microscopy). Raman microscopy significantly overestimated the degree of cure and failed to provide reliable information for the salt yield in the glass ionomer cement. Fourier transform infrared microscopy showed increased, but no statistically significant difference in, aluminum-carboxylate salts in the retrieved specimens. CONCLUSIONS: Enhanced oxidation of residual carbon-carbon bonds in the composite and slightly increased dissolution of the weaker calcium-salt phase in the glass ionomer cement were the main differences in the intraorally aged specimens in comparison with the specimens stored in water.

Is Diet a Determining Factor in the Induction of Gingival Inflammation by Dental Plaque? A Secondary Analysis of Clinical Studies.

The aim was to determine the association between plaque and gingival inflammation reported by dietary interventions. Data of four clinical studies dealing with changed nutrition and gingival examination were reanalyzed with regard to gingival inflammation (GI), plaque (PI), and bleeding on probing (BOP). Dietary changes basically involved avoiding sugar, white flour and sweetened drinks and focusing on whole foods for 4 weeks. The control groups were to maintain their usual diet. All participants had to reduce their oral hygiene efforts. Linear regression models taking the clustering of the data due to several studies into account were applied. In total, data of 92 participants (control groups: 39, test-groups 53) were reanalyzed. While both groups showed a slight increase in dental plaque, only the test groups showed a significant decrease in inflammatory parameters: GI (mean value difference End-Baseline (Δ): -0.31 (±SD 0.36)) and BOP (Δ: -15.39% (±16.07)), both p < 0.001. In the control groups, there was a constant relation between PI and GI, while the experimental group showed a decreasing relationship in GI/PI (p = 0.016), and even an inverted relationship BOP/PI under a changed diet (p = 0.031). In conclusion, diet seems to be a determining factor how the gingiva reacts towards dental plaque.

Predictive simulations and optimization of nanowire field-effect PSA sensors including screening.

We apply our self-consistent PDE model for the electrical response of field-effect sensors to the 3D simulation of nanowire PSA (prostate-specific antigen) sensors. The charge concentration in the biofunctionalized boundary layer at the semiconductor-electrolyte interface is calculated using the propka algorithm, and the screening of the biomolecules by the free ions in the liquid is modeled by a sensitivity factor. This comprehensive approach yields excellent agreement with experimental current-voltage characteristics without any fitting parameters. Having verified the numerical model in this manner, we study the sensitivity of nanowire PSA sensors by changing device parameters, making it possible to optimize the devices and revealing the attributes of the optimal field-effect sensor.

Kinetic parameter estimation and fluctuation analysis of CO at SnO2 single nanowires.

In this work, we present calculated numerical values for the kinetic parameters governing adsorption/desorption processes of carbon monoxide at tin dioxide single-nanowire gas sensors. The response of such sensors to pulses of 50 ppm carbon monoxide in nitrogen is investigated at different temperatures to extract the desired information. A rate-equation approach is used to model the reaction kinetics, which results in the problem of determining coefficients in a coupled system of nonlinear ordinary differential equations. The numerical values are computed by inverse-modeling techniques and are then used to simulate the sensor response. With our model, the dynamic response of the sensor due to the gas-surface interaction can be studied in order to find the optimal setup for detection, which is an important step towards selectivity of these devices. We additionally investigate the noise in the current through the nanowire and its changes due to the presence of carbon monoxide in the sensor environment. Here, we propose the use of a wavelet transform to decompose the signal and analyze the noise in the experimental data. This method indicates that some fluctuations are specific for the gas species investigated here.

Expression analysis of a family of developmentally-regulated cytosolic sulfotransferases (SULTs) in Drosophila.

We have compared the amino acid sequence of all four cytosolic sulfotransferases (SULTs) in Drosophila and analyzed their spatial expression patterns during development. Three out of four SULTs show distinct expression activity during embryogenesis, while the 4th SULT shows expression only post-embryonically. st1, st3 and st4 are expressed in non-overlapping expression domains mainly confined to organs of the alimentary canal such as esophagus, malphigian tubules, hindgut, as well as in the tracheal system. All these organs are surrounded by the hemolymph suggesting that Drosophila SULTs exert their function in detoxification of substances upon influx from the hemolymph.

chinmo-mutant spermatogonial stem cells cause mitotic drive by evicting non-mutant neighbors from the niche.

Niches maintain a finite pool of stem cells via restricted space and short-range signals. Stem cells compete for limited niche resources, but the mechanisms regulating competition are poorly understood. Using the Drosophila testis model, we show that germline stem cells (GSCs) lacking the transcription factor Chinmo gain a competitive advantage for niche access. Surprisingly, chinmo-/- GSCs rely on a new mechanism of competition in which they secrete the extracellular matrix protein Perlecan to selectively evict non-mutant GSCs and then upregulate Perlecan-binding proteins to remain in the altered niche. Over time, the GSC pool can be entirely replaced with chinmo-/- cells. As a consequence, the mutant chinmo allele acts as a gene drive element; the majority of offspring inherit the allele despite the heterozygous genotype of the parent. Our results suggest that the influence of GSC competition may extend beyond individual stem cell niche dynamics to population-level allelic drift and evolution.

Anti-Fibrotic Activity of an Antimicrobial Peptide in a Drosophila Model.

Fibrotic lesions accompany several pathological conditions, including tumors. We show that expression of a dominant-active form of the Ras oncogene in Drosophila salivary glands (SGs) leads to redistribution of components of the basement membrane (BM) and fibrotic lesions. Similar to several types of mammalian fibrosis, the disturbed BM attracts clot components, including insect transglutaminase and phenoloxidase. SG epithelial cells show reduced apicobasal polarity accompanied by a loss of secretory activity. Both the fibrotic lesions and the reduced cell polarity are alleviated by ectopic expression of the antimicrobial peptide drosomycin (Drs), which also restores the secretory activity of the SGs. In addition to extracellular matrix components, both Drs and F-actin localize to fibrotic lesions.

Quantification reveals early dynamics in Drosophila maternal gradients.

The Bicoid (Bcd) protein is a primary determinant of early anterior-posterior (AP) axis specification in Drosophila embryogenesis. This morphogen is spatially distributed in an anterior-high gradient, and affects particular AP cell fates in a concentration-dependent manner. The early distribution and dynamics of the bicoid (bcd) mRNA, the source for the Bcd protein gradient, is not well understood, leaving a number of open questions for how Bcd positional information develops and is regulated. Confocal microscope images of whole early embryos, stained for bcd mRNA or the Staufen (Stau) protein involved in its transport, were processed to extract quantitative AP intensity profiles at two depths (apical-under the embryo surface but above the nuclear layer; and basal-below the nuclei). Each profile was quantified by a two- (or three-) exponential equation. The parameters of these equations were used to analyze the early developmental dynamics of bcd. Analysis of 1D profiles was compared with 2D intensity surfaces from the same images. This approach reveals strong early changes in bcd and Stau, which appear to be coordinated. We can unambiguously discriminate three stages in early development using the exponential parameters: pre-blastoderm (1-9 cleavage cycle, cc), syncytial blastoderm (10-13 cc) and cellularization (from 14A cc). Key features which differ in this period are how fast the first exponential (anterior component) of the apical profile drops with distance and whether it is higher or lower than the basal first exponential. We can further discriminate early and late embryos within the pre-blastoderm stage, depending on how quickly the anterior exponential drops. This relates to the posterior-wards spread of bcd in the first hour of development. Both bcd and Stau show several redistributions in the head cytoplasm, quite probably related to nuclear activity: first shifting inwards towards the core plasm, forming either protrusions (early pre-blastoderm) or round aggregations (early nuclear cleavage cycles, cc, 13 and 14), then moving to the embryo surface and spreading posteriorly. These movements are seen both with the 2D surface study and the 1D profile analysis. The continued spreading of bcd can be tracked from the time of nuclear layer formation (later pre-blastoderm) to the later syncytial blastoderm stages by the progressive loss of steepness of the apical anterior exponential (for both bcd and Stau). Finally, at the beginning of cc14 (cellularization stage) we see a distinctive flip from the basal anterior gradient being higher to the apical gradient being higher (for both bcd and Stau). Quantitative analysis reveals substantial (and correlated) bcd and Stau redistributions during early development, supporting that the distribution and dynamics of bcd mRNA are key factors in the formation and maintenance of the Bcd protein morphogenetic gradient. This analysis reveals the complex and dynamic nature of bcd redistribution, particularly in the head cytoplasm. These resemble observations in oogenesis; their role and significance have yet to be clarified. The observed co-localization during redistribution of bcd and Stau may indicate the involvement of active transport.