Early Drosophila Development Research Articles

Steroid Signaling within Drosophila Ovarian Epithelial Cells Sex-Specifically Modulates Early Germ Cell Development and Meiotic Entry

Drosophila adult females but not males contain high levels of the steroid hormone ecdysone, however, the roles played by steroid signaling during Drosophila gametogenesis remain poorly understood. Drosophila germ cells in both sexes initially follow a similar pathway. After germline stem cells are established, their daughters form interconnected cysts surrounded by somatic escort (female) or cyst (male) cells and enter meiosis. Subsequently, female cysts acquire a new covering of somatic cells to form follicles. Knocking down expression of the heterodimeric ecdysteroid receptor (EcR/Usp) or the E75 early response gene in escort cells disrupts 16-cell cyst production, meiotic entry and follicle formation. Escort cells lose their squamous morphology and unsheath germ cells. By contrast, disrupting ecdysone signaling in males does not perturb cyst development or ensheathment. Thus, sex-specific steroid signaling is essential for female germ cell development at the time male and female pathways diverge. Our results suggest that steroid signaling plays an important sex-specific role in early germ cell development in Drosophila, a strategy that may be conserved in mammals.

A parallel multi-objective optimization algorithm for the calibration of mathematical models

Based on evolutionary computation techniques, we present a parallel, globally convergent, multi-objective optimization algorithm which extends the Covariance Matrix Adaptation Evolutionary Strategy (CMA-ES). This approach enables identifying multiple global optima and multiple discontinuous Pareto set solutions of the optimization problem in a compact search space. After evaluating the algorithm with test functions, we apply our method to the identification of the parameters of a reaction–diffusion model of a genetic regulatory mechanism during Drosophila early development, our simulations being in agreement with the experimental data. Comparisons with a multi-objective version of the CMA-ES (MO-CMA-ES) on this dataset show that our algorithm outperforms largely the speed of convergence of MO-CMA-ES. We have identified an infinite number of accurate solutions of the model equations, associated with the Pareto set of the optimization problem. This non-unicity property of a biological developmental process explains phenotypic plasticity and resilience in biological systems.

Crystal structure of a minimal eIF4E–Cup complex reveals a general mechanism of eIF4E regulation in translational repression

Cup is an eIF4E-binding protein (4E-BP) that plays a central role in translational regulation of localized mRNAs during early Drosophila development. In particular, Cup is required for repressing translation of the maternally contributed oskar, nanos, and gurken mRNAs, all of which are essential for embryonic body axis determination. Here, we present the 2.8 Å resolution crystal structure of a minimal eIF4E-Cup assembly, consisting of the interacting regions of the two proteins. In the structure, two separate segments of Cup contact two orthogonal faces of eIF4E. The eIF4E-binding consensus motif of Cup (YXXXXLΦ) binds the convex side of eIF4E similarly to the consensus of other eIF4E-binding proteins, such as 4E-BPs and eIF4G. The second, noncanonical, eIF4E-binding site of Cup binds laterally and perpendicularly to the eIF4E β-sheet. Mutations of Cup at this binding site were shown to reduce binding to eIF4E and to promote the destabilization of the associated mRNA. Comparison with the binding mode of eIF4G to eIF4E suggests that Cup and eIF4G binding would be mutually exclusive at both binding sites. This shows how a common molecular surface of eIF4E might recognize different proteins acting at different times in the same pathway. The structure provides insight into the mechanism by which Cup disrupts eIF4E-eIF4G interaction and has broader implications for understanding the role of 4E-BPs in translational regulation.

Developmental expression ofDrosophilaWiskott‐Aldrich Syndrome family proteins

Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics.

Doubling the Dose

Doubling the Dose

Quantitative models of the mechanisms that control genome-wide patterns of transcription factor binding during early Drosophila development.

Transcription factors that drive complex patterns of gene expression during animal development bind to thousands of genomic regions, with quantitative differences in binding across bound regions mediating their activity. While we now have tools to characterize the DNA affinities of these proteins and to precisely measure their genome-wide distribution in vivo, our understanding of the forces that determine where, when, and to what extent they bind remains primitive. Here we use a thermodynamic model of transcription factor binding to evaluate the contribution of different biophysical forces to the binding of five regulators of early embryonic anterior-posterior patterning in Drosophila melanogaster. Predictions based on DNA sequence and in vitro protein-DNA affinities alone achieve a correlation of ∼0.4 with experimental measurements of in vivo binding. Incorporating cooperativity and competition among the five factors, and accounting for spatial patterning by modeling binding in every nucleus independently, had little effect on prediction accuracy. A major source of error was the prediction of binding events that do not occur in vivo, which we hypothesized reflected reduced accessibility of chromatin. To test this, we incorporated experimental measurements of genome-wide DNA accessibility into our model, effectively restricting predicted binding to regions of open chromatin. This dramatically improved our predictions to a correlation of 0.6–0.9 for various factors across known target genes. Finally, we used our model to quantify the roles of DNA sequence, accessibility, and binding competition and cooperativity. Our results show that, in regions of open chromatin, binding can be predicted almost exclusively by the sequence specificity of individual factors, with a minimal role for protein interactions. We suggest that a combination of experimentally determined chromatin accessibility data and simple computational models of transcription factor binding may be used to predict the binding landscape of any animal transcription factor with significant precision.

Retrotransposition of R2 elements in somatic nuclei during the early development of Drosophila

BackgroundR2 retrotransposable elements exclusively insert in the 28S rRNA genes of their host. Their RNA transcripts are produced by self-processing from a 28S R2 cotranscript. Because full-length R2 transcripts are found in most tissues of R2-active animals, we tested whether new R2 insertions occurred in somatic tissues even though such events would be an evolutionary dead end.FindingsPCR assays were used to identify somatic R2 insertions in isolated adult tissues and larval imaginal discs of Drosophila simulans. R2 somatic mosaics were detected encompassing cells from individual tissues as well as tissues from multiple body segments. The somatic insertions had 5' junction sequences characteristic of germline insertions suggesting they represented authentic retrotransposition events.ConclusionsBody segments are specified early in Drosophila development, thus the detection of the same somatic insertion in cells from multiple tissues suggested that the R2 retrotransposition events had occurred before the blastoderm stage of Drosophila development. R2 activity at this stage, when embryonic nuclei are rapidly dividing in a common cytoplasm, suggests that some retrotransposition events appearing as germline events may correspond to germline mosaicism.

Calibration and validation of a genetic regulatory network model describing the production of the protein Hunchback in Drosophila early development

We fit the parameters of a differential equations model describing the production of gap-gene proteins Hunchback and Knirps along the antero-posterior axis of the embryo of Drosophila. As initial data for the differential equations model, we take the antero-posterior distribution of the proteins Bicoid, Hunchback and Tailless at the beginning of cleavage cycle 14. We calibrate and validate the model with experimental data using single- and multi-objective evolutionary optimization techniques. In the multi-objective optimization technique, we compute the associated Pareto fronts. We analyze the cross regulation mechanism between the gap-genes protein pair Hunchback-Knirps and we show that the posterior distribution of Hunchback follow the experimental data if Hunchback is negatively regulated by the Huckebein protein. This approach enables to us predict the posterior localization on the embryo of the protein Huckebein, and to validate with the experimental data the genetic regulatory network responsible for the antero-posterior distribution of the gap-gene protein Hunchback. We discuss the importance of Pareto multi-objective optimization techniques in the calibration and validation of biological models.

Wnt/β-catenin dependent cell proliferation underlies segmented lateral line morphogenesis

Morphogenesis is a fascinating but complex and incompletely understood developmental process. The sensory lateral line system consists of only a few hundred cells and is experimentally accessible making it an excellent model system to interrogate the cellular and molecular mechanisms underlying segmental morphogenesis. The posterior lateral line primordium periodically deposits prosensory organs as it migrates to the tail tip. We demonstrate that periodic proneuromast deposition is governed by a fundamentally different developmental mechanism than the classical models of developmental periodicity represented by vertebrate somitogenesis and early Drosophila development. Our analysis demonstrates that proneuromast deposition is driven by periodic lengthening of the primordium and a stable Wnt/β-catenin activation domain in the leading region of the primordium. The periodic lengthening of the primordium is controlled by Wnt/β-catenin/Fgf-dependent proliferation. Once proneuromasts are displaced into the trailing Wnt/β-catenin-free zone they are deposited. We have previously shown that Wnt/β-catenin signaling induces Fgf signaling and that interactions between these two pathways regulate primordium migration and prosensory organ formation. Therefore, by coordinating migration, prosensory organ formation and proliferation, localized activation of Wnt/β-catenin signaling in the leading zone of the primordium plays a crucial role in orchestrating lateral line morphogenesis.

Wg signaling in Drosophila heart development as a pioneering model

The heart is one of the first functional embryonic organs occurring during development. The fundamental developmental processes and genes involved in cardiogenesis are conserved between the invertebrates and vertebrates. In the past fifteen years, one of signaling pathways that has been best characterized in heart development in both invertebrates and vertebrates is the Wg/Wnt signaling pathways. Since our discovery of the Wg signaling required for the early heart development in Drosophila, the past fifteen years have witnessed tremendous progress in the understanding of specific Wnt signaling pathways in vertebrate cardiogenesis. This review will summarize the current state of knowledge of Wg signaling transduction in Drosophila heart development, which will benefit our understanding of vertebrate cardiogenesis and human congenital malformations.

A Software Tool to Model Genetic Regulatory Networks. Applications to the Modeling of Threshold Phenomena and of Spatial Patterning in Drosophila

We present a general methodology in order to build mathematical models of genetic regulatory networks. This approach is based on the mass action law and on the Jacob and Monod operon model. The mathematical models are built symbolically by the Mathematica software package GeneticNetworks. This package accepts as input the interaction graphs of the transcriptional activators and repressors of a biological process and, as output, gives the mathematical model in the form of a system of ordinary differential equations. All the relevant biological parameters are chosen automatically by the software. Within this framework, we show that concentration dependent threshold effects in biology emerge from the catalytic properties of genes and its associated conservation laws. We apply this methodology to the segment patterning in Drosophila early development and we calibrate the genetic transcriptional network responsible for the patterning of the gap gene proteins Hunchback and Knirps, along the antero-posterior axis of the Drosophila embryo. In this approach, the zygotically produced proteins Hunchback and Knirps do not diffuse along the antero-posterior axis of the embryo of Drosophila, developing a spatial pattern due to concentration dependent thresholds. This shows that patterning at the gap genes stage can be explained by the concentration gradients along the embryo of the transcriptional regulators.

Stationary and oscillatory fronts in a two-component genetic regulatory network model

We investigate a two-component gene network model, originally used to describe the spatiotemporal patterning of the gene products in early Drosophila development. By considering a particular mode of interaction between the two gene products, denoted proteins A and B , we find both stable stationary and time-oscillatory fronts can occur in the reaction–diffusion system. We reduce the system by replacing B with its spatial average (shadow system) and assume an abrupt “on-and-off” switch for the genes. In doing so, explicit formula are obtained for all steady-state solutions and their linear eigenvalues. Using the diffusion of A , D a , and the basal production rate, r , as bifurcation parameters, we explore ranges in which a monotone, stationary front is stable, and show it can lose stability through a Hopf bifurcation, giving rise to oscillatory fronts. We also discuss the existence and stability of steady-state and time-oscillatory solutions with multiple extrema. An intuitive explanation for the occurrence of stable stationary and oscillatory front solutions is provided based on the behavior of A in the absence of B and the opposite regulation between A and B . Such behavior is also interpreted in terms of the biological parameters in the model, including those governing the connection of the gene network.

MRNA diffusion explains protein gradients in Drosophila early development

We propose a new model describing the production and the establishment of the stable gradient of the Bicoid protein along the antero-posterior axis of the embryo of Drosophila. In this model, we consider that bicoid mRNA diffuses along the antero-posterior axis of the embryo and the protein is produced in the ribosomes localized near the syncytial nuclei. Bicoid protein stays localized near the syncytial nuclei as observed in experiments. We calibrate the parameters of the mathematical model with experimental data taken during the cleavage stages 11–14 of the developing embryo of Drosophila. We obtain good agreement between the experimental and the model gradients, with relative errors in the range 5–8%. The inferred diffusion coefficient of bicoid mRNA is in the range 4.6 × 10 − 12 − 1.5 × 10 − 11 m 2 s − 1 , in agreement with the theoretical predictions and experimental measurements for the diffusion of macromolecules in the cytoplasm. We show that the model based on the mRNA diffusion hypothesis is consistent with the known observational data, supporting the recent experimental findings of the gradient of bicoid mRNA in Drosophila [Spirov et al. (2009). Development 136, 605–614].

Genome-wide analysis of mRNA decay patterns during early Drosophiladevelopment

BackgroundThe modulation of mRNA levels across tissues and time is key for the establishment and operation of the developmental programs that transform the fertilized egg into a fully formed embryo. Although the developmental mechanisms leading to differential mRNA synthesis are heavily investigated, comparatively little attention is given to the processes of mRNA degradation and how these relate to the molecular programs controlling development.ResultsHere we combine timed collection of Drosophila embryos and unfertilized eggs with genome-wide microarray technology to determine the degradation patterns of all mRNAs present during early fruit fly development. Our work studies the kinetics of mRNA decay, the contributions of maternally and zygotically encoded factors to mRNA degradation, and the ways in which mRNA decay profiles relate to gene function, mRNA localization patterns, translation rates and protein turnover. We also detect cis-regulatory sequences enriched in transcripts with common degradation patterns and propose several proteins and microRNAs as developmental regulators of mRNA decay during early fruit fly development. Finally, we experimentally validate the effects of a subset of cis-regulatory sequences and trans-regulators in vivo.ConclusionsOur work advances the current understanding of the processes controlling mRNA degradation during early Drosophila development, taking us one step closer to the understanding of mRNA decay processes in all animals. Our data also provide a valuable resource for further experimental and computational studies investigating the process of mRNA decay.

MRNA diffusion explains protein gradients in Drosophila early development

AbstractWe propose a new model describing the production and the establishment of the stable gradient of the Bicoid protein along the antero-posterior axis of the embryo of Drosophila. In this model, we consider that bicoid mRNA diffuses along the antero-posterior axis of the embryo and the protein is produced in the ribosomes localized near the syncytial nuclei. Bicoid protein stays localized near the syncytial nuclei as observed in experiments.We calibrate the parameters of the mathematical model with experimental data taken during the cleavage stages 11 to 14 of the developing embryo of Drosophila. We obtain good agreement between the experimental and the model gradients, with relative errors in the range 5-8%. The inferred diffusion coefficient of bicoid mRNA is in the range 4.6 x 10^-12^ - 1.5 x10^-11^ m^2^s^-1^, in agreement with the theoretical predictions and experimental measurements for the diffusion of macromolecules in the cytoplasm. We show that the model based on the mRNA diffusion hypothesis is consistent with the known observational data, supporting the recent experimental findings of the gradient of bicoid mRNA in Drosophila [Spirov et al. (2009) Development 136:605-614].

How I became a biochemist

How I became a biochemist

Abundant genetic variation in transcript level during early Drosophila development.

Variation in gene expression may underlie many important evolutionary traits. However, it is not known at what stage in organismal development changes in gene expression are most likely to result in changes in phenotype. One widely held belief is that changes in early development are more likely to result in changes in downstream phenotypes. In order to discover how much genetic variation for transcript level is present in natural populations, we studied zygotic gene expression in nine inbred lines of Drosophila melanogaster at two time points in their development. We find abundant variation for transcript level both between lines and over time; close to half of all expressed genes show a significant line effect at either time point. We examine the contribution of maternally loaded genes to this variation, as well as the contribution of variation in upstream genes to variation in their downstream targets in two well-studied gene regulatory networks. Finally, we estimate the dimensionality of gene expression in these two networks and find that-despite large numbers of varying genes-there appear to be only two factors controlling this variation.

Microevolutionary support for a developmental hourglass: gene expression patterns shape sequence variation and divergence inDrosophila

A central goal of evolutionary developmental biology (Evo-Devo) is to synthesize comparative molecular developmental genetics and its description of the dynamic relationship between genotype and phenotype with the microevolutionary processes (mutation, random drift, and selection) of population genetics. To this end, we analyzed sequence variation of five gene classes that act sequentially to shape early embryo development in Drosophila: maternal, gap, pair-rule, segment polarity, and segment identity genes. We found two related patterns: (1) a microevolutionary pattern, wherein relative sequence variation within species is 2- to 3-fold higher for maternal-effect genes than for any other gene class; and, (2) a macroevolutionary pattern, wherein the relative sequence divergence among species for maternal-effect genes is 2- to 4-fold greater than for any other gene class. Both patterns are qualitatively and quantitatively consistent with the predictions of microevolutionary theory. Our findings connect within-species genetic variation to between-species divergence and shed light on the controversy over the existence of a "developmental hourglass," where mid-embryonic stages are more evolutionarily constrained than either earlier or later stages. Because maternal-effect genes experience relaxed selective constraint relative to zygotic-effect genes, they explore a wider mutational and phenotypic space. As a result, early acting maternal-effect genes diverge more widely across taxa and thereby broaden the base of the developmental hourglass. In contrast, later acting zygotic genes are relatively more constrained and limited in their diversification across taxa, narrowing the waist of the developmental hourglass. This pattern is obscured by genes with both maternal and zygotic expression, which experience the strongest evolutionary constraint.

Anillin-mediated Targeting of Peanut to Pseudocleavage Furrows Is Regulated by the GTPase Ran

During early development in Drosophila, pseudocleavage furrows in the syncytial embryo prevent contact between neighboring spindles, thereby ensuring proper chromosome segregation. Here we demonstrate that the GTPase Ran regulates pseudocleavage furrow organization. Ran can exert control on pseudocleavage furrows independently of its role in regulating the microtubule cytoskeleton. Disruption of the Ran pathway prevented pseudocleavage furrow formation and restricted the depth and duration of furrow ingression of those pseudocleavage furrows that did form. We found that Ran was required for the localization of the septin Peanut to the pseudocleavage furrow, but not anillin or actin. Biochemical assays revealed that the direct binding of the nuclear transport receptors importin alpha and beta to anillin prevented the binding of Peanut to anillin. Furthermore, RanGTP reversed the inhibitory action of importin alpha and beta. On expression of a mutant form of anillin that lacked an importin alpha and beta binding site, inhibition of Ran no longer restricted the depth and duration of furrow ingression in those pseudocleavage furrows that formed. These data suggest that anillin and Peanut are involved in pseudocleavage furrow ingression in syncytial embryos and that this process is regulated by Ran.

Optimizing the readout of morphogen gradients

In multicellular organisms, the initial patterns of gene expression are regulated by spatial gradients of biochemical factors, known as morphogen gradients. Because of biochemical noise in the morphogen gradients there are associated spatial errors in the positions of target gene patterns. Using a simple single morphogen and/or single target gene model, we use propagation of error analysis to derive a condition on the amount of morphogen that needs to be produced in order to have precise spatial patterning of the target. We find that there is an optimal morphogen gradient profile that requires the least amount of morphogen to be produced. Experimental results for the Bicoid-Hunchback system in early Drosophila development are consistent with the predictions of this analysis. We also discuss our results in the context of recent work that analyzed this system using mutual information as an organizing principle, and show that minimizing the amount of morphogen produced also leads to a near optimal flow of information between input and target.