Publications

2017
Falahati, Hanieh, and Eric Wieschaus. “Independent active and thermodynamic processes govern the nucleolus assembly in vivo.”. Proc Natl Acad Sci U S A 114.6 (2017): , 114, 6, 1335-1340. Web.Abstract
Membraneless organelles play a central role in the organization of protoplasm by concentrating macromolecules, which allows efficient cellular processes. Recent studies have shown that, in vitro, certain components in such organelles can assemble through phase separation. Inside the cell, however, such organelles are multicomponent, with numerous intermolecular interactions that can potentially affect the demixing properties of individual components. In addition, the organelles themselves are inherently active, and it is not clear how the active, energy-consuming processes that occur constantly within such organelles affect the phase separation behavior of the constituent macromolecules. Here, we examine the phase separation model for the formation of membraneless organelles in vivo by assessing the two features that collectively distinguish it from active assembly, namely temperature dependence and reversibility. We use a microfluidic device that allows accurate and rapid manipulation of temperature and examine the quantitative dynamics by which six different nucleolar proteins assemble into the nucleoli of Drosophila melanogaster embryos. Our results indicate that, although phase separation is the main mode of recruitment for four of the studied proteins, the assembly of the other two is irreversible and enhanced at higher temperatures, behaviors indicative of active recruitment to the nucleolus. These two subsets of components differ in their requirements for ribosomal DNA; the two actively assembling components fail to assemble in the absence of ribosomal DNA, whereas the thermodynamically driven components assemble but lose temporal and spatial precision.
Doubrovinski, Konstantin, et al.Measurement of cortical elasticity in Drosophila melanogaster embryos using ferrofluids.”. Proc Natl Acad Sci U S A 114.5 (2017): , 114, 5, 1051-1056. Web.Abstract
Many models of morphogenesis are forced to assume specific mechanical properties of cells, because the actual mechanical properties of living tissues are largely unknown. Here, we measure the rheology of epithelial cells in the cellularizing Drosophila embryo by injecting magnetic particles and studying their response to external actuation. We establish that, on timescales relevant to epithelial morphogenesis, the cytoplasm is predominantly viscous, whereas the cellular cortex is elastic. The timescale of elastic stress relaxation has a lower bound of 4 min, which is comparable to the time required for internalization of the ventral furrow during gastrulation. The cytoplasm was measured to be ∼10(3)-fold as viscous as water. We show that elasticity depends on the actin cytoskeleton and conclude by discussing how these results relate to existing mechanical models of morphogenesis.
Weng, Mo, and Eric Wieschaus. “Polarity protein Par3/Bazooka follows myosin-dependent junction repositioning.”. Dev Biol 422.2 (2017): , 422, 2, 125-134. Web.Abstract
The polarity protein Par3/Bazooka (Baz) has been established as a central component of the apical basal polarity system that determines the position of cell-cell junctions in epithelial cells. Consistent with that view, we show that shortly before gastrulation in Drosophila, Baz protein in the mesoderm is down-regulated from junctional sites in response to Snail (Sna) expression. This down-regulation leads to a specific decrease in adherens junctions without affecting other E-Cadherin pools. However, we further show that, interactions between Baz and junctions are not unidirectional. During apical constriction and the internalization of the mesoderm, down-regulation of Baz is transiently blocked as adherens junctions shift apically and are strengthened in response to tension generated by contractile actomyosin. When such junction remodeling is prevented by down-regulating myosin, Baz is lost prematurely in mesodermal epithelium. During such apical shifts, Baz is initially left behind as the junction shifts position, but then re-accumulates at the new location of the junctions. On the dorsal side of the embryo, a similar pattern of myosin activity appears to limit the basal shift in junctions normally driven by Baz that controls epithelium folding. Our results suggest a model where the sensitivity of Baz to Sna expression leads to the Sna-dependent junction disassembly required for a complete epithelium-mesenchymal transition. Meanwhile this loss of Baz-dependent junction maintenance is countered by the myosin-based mechanism which promotes an apical shift and strengthening of junctions accompanied by a transient re-positioning and maintenance of Baz proteins.
2016
Blythe, Shelby A, and Eric F Wieschaus. “Establishment and maintenance of heritable chromatin structure during early Drosophila embryogenesis.”. Elife 5 (2016). Web.Abstract
During embryogenesis, the initial chromatin state is established during a period of rapid proliferative activity. We have measured with 3-min time resolution how heritable patterns of chromatin structure are initially established and maintained during the midblastula transition (MBT). We find that regions of accessibility are established sequentially, where enhancers are opened in advance of promoters and insulators. These open states are stably maintained in highly condensed mitotic chromatin to ensure faithful inheritance of prior accessibility status across cell divisions. The temporal progression of establishment is controlled by the biological timers that control the onset of the MBT. In general, acquisition of promoter accessibility is controlled by the biological timer that measures the nucleo-cytoplasmic (N:C) ratio, whereas timing of enhancer accessibility is regulated independently of the N:C ratio. These different timing classes each associate with binding sites for two transcription factors, GAGA-factor and Zelda, previously implicated in controlling chromatin accessibility at ZGA.
He, Bing, Adam Martin, and Eric Wieschaus. “Flow-dependent myosin recruitment during Drosophila cellularization requires zygotic dunk activity.”. Development 143.13 (2016): , 143, 13, 2417-30. Web.Abstract
Actomyosin contractility underlies force generation in morphogenesis ranging from cytokinesis to epithelial extension or invagination. In Drosophila, the cleavage of the syncytial blastoderm is initiated by an actomyosin network at the base of membrane furrows that invaginate from the surface of the embryo. It remains unclear how this network forms and how it affects tissue mechanics. Here, we show that during Drosophila cleavage, myosin recruitment to the cleavage furrows proceeds in temporally distinct phases of tension-driven cortical flow and direct recruitment, regulated by different zygotic genes. We identify the gene dunk, which we show is transiently transcribed when cellularization starts and functions to maintain cortical myosin during the flow phase. The subsequent direct myosin recruitment, however, is Dunk-independent but requires Slam. The Slam-dependent direct recruitment of myosin is sufficient to drive cleavage in the dunk mutant, and the subsequent development of the mutant is normal. In the dunk mutant, cortical myosin loss triggers misdirected flow and disrupts the hexagonal packing of the ingressing furrows. Computer simulation coupled with laser ablation suggests that Dunk-dependent maintenance of cortical myosin enables mechanical tension build-up, thereby providing a mechanism to guide myosin flow and define the hexagonal symmetry of the furrows.
Wieschaus, Eric, and Christiane Nüsslein-Volhard. “The Heidelberg Screen for Pattern Mutants of Drosophila: A Personal Account.”. Annu Rev Cell Dev Biol 32 (2016): , 32, 1-46. Web.Abstract
In large-scale mutagenesis screens performed in 1979-1980 at the EMBL in Heidelberg, we isolated mutations affecting the pattern or structure of the larval cuticle in Drosophila. The 600 mutants we characterized could be assigned to 120 genes and represent the majority of such genes in the genome. These mutants subsequently provided a rich resource for understanding many fundamental developmental processes, such as the transcriptional hierarchies controlling segmentation, the establishment of cell states by signaling pathways, and the differentiation of epithelial cells. Most of the Heidelberg genes are now molecularly known, and many of them are conserved in other animals, including humans. Although the screens were initially driven entirely by curiosity, the mutants now serve as models for many human diseases. In this review, we describe the rationale of the screening procedures and provide a classification of the genes on the basis of their initial phenotypes and the subsequent molecular analyses.
Weng, Mo, and Eric Wieschaus. “Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly.”. J Cell Biol 212.2 (2016): , 212, 2, 219-29. Web.Abstract
Although Snail is essential for disassembly of adherens junctions during epithelial-mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, we track the behavior of E-cadherin-rich junction clusters, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. Our findings provide in vivo evidence for mechanosensitivity of cell-cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT.
Falahati, Hanieh, et al.Nucleation by rRNA Dictates the Precision of Nucleolus Assembly.”. Curr Biol 26.3 (2016): , 26, 3, 277-85. Web.Abstract
Membrane-less organelles are intracellular compartments specialized to carry out specific cellular functions. There is growing evidence supporting the possibility that such organelles form as a new phase, separating from cytoplasm or nucleoplasm. However, a main challenge to such phase separation models is that the initial assembly, or nucleation, of the new phase is typically a highly stochastic process and does not allow for the spatiotemporal precision observed in biological systems. Here, we investigate the initial assembly of the nucleolus, a membrane-less organelle involved in different cellular functions including ribosomal biogenesis. We demonstrate that the nucleolus formation is precisely timed in D. melanogaster embryos and follows the transcription of rRNA. We provide evidence that transcription of rRNA is necessary for overcoming the highly stochastic nucleation step in the formation of the nucleolus, through a seeding mechanism. In the absence of rDNA, the nucleolar proteins studied are able to form high-concentration assemblies. However, unlike the nucleolus, these assemblies are highly variable in number, location, and time at which they form. In addition, quantitative study of the changes in the nucleoplasmic concentration and distribution of these nucleolar proteins in the wild-type embryos is consistent with the role of rRNA in seeding the nucleolus formation.
Wieschaus, Eric. “Positional Information and Cell Fate Determination in the Early Drosophila Embryo.”. Curr Top Dev Biol 117 (2016): , 117, 567-79. Web.Abstract
During early development in Drosophila, the spatial information of maternal gradients is translated into discrete transcriptional states determining cell fate. Information transfer depends on reproducibility of the gradients themselves, as well as the ability of cells to accurately measure and utilize morphogen concentrations in biologically meaning ways. In the following essay, these issues are discussed in context of the Bicoid gradient.
Momen-Roknabadi, Amir, Stefano Di Talia, and Eric Wieschaus. “Transcriptional Timers Regulating Mitosis in Early Drosophila Embryos.”. Cell Rep 16.11 (2016): , 16, 11, 2793-801. Web.Abstract
The development of an embryo requires precise spatiotemporal regulation of cellular processes. During Drosophila gastrulation, a precise temporal pattern of cell division is encoded through transcriptional regulation of cdc25(string) in 25 distinct mitotic domains. Using a genetic screen, we demonstrate that the same transcription factors that regulate the spatial pattern of cdc25(string) transcription encode its temporal activation. We identify buttonhead and empty spiracles as the major activators of cdc25(string) expression in mitotic domain 2. The effect of these activators is balanced through repression by hairy, sloppy paired 1, and huckebein. Within the mitotic domain, temporal precision of mitosis is robust and unaffected by changing dosage of rate-limiting transcriptional factors. However, precision can be disrupted by altering the levels of the two activators or two repressors. We propose that the additive and balanced action of activators and repressors is a general strategy for precise temporal regulation of cellular transitions during development.
2015
Blythe, Shelby A, and Eric F Wieschaus. “Coordinating Cell Cycle Remodeling with Transcriptional Activation at the Drosophila MBT.”. Curr Top Dev Biol 113 (2015): , 113, 113-48. Web.Abstract
During the maternal-to-zygotic transition (MZT), major changes in cell cycle regulation coincide with large-scale zygotic genome activation. In this chapter, we discuss the current understanding of how the cell cycle is remodeled over the course of the Drosophila MZT, and how the temporal precision of this event is linked to contemporaneous alterations in genome-wide chromatin structure and transcriptional activity. The cell cycle is initially lengthened during the MZT by activation of the DNA replication checkpoint but, subsequently, zygotically supplied factors are essential for establishing lasting modifications to the cell cycle.
Osterfield, Miriam, et al.Diversity of epithelial morphogenesis during eggshell formation in drosophilids.”. Development 142.11 (2015): , 142, 11, 1971-7. Web.Abstract
The eggshells of drosophilid species provide a powerful model for studying the origins of morphological diversity. The dorsal appendages, or respiratory filaments, of these eggshells display a remarkable interspecies variation in number and shape, and the epithelial patterning underlying the formation of these structures is an area of active research. To extend the analysis of dorsal appendage formation to include morphogenesis, we developed an improved 3D image reconstruction approach. This approach revealed considerable interspecies variation in the cell shape changes and neighbor exchanges underlying appendage formation. Specifically, although the appendage floor in Drosophila melanogaster is formed through spatially ordered neighbor exchanges, the same structure in Scaptodrosophila pattersoni is formed through extreme changes in cell shape, whereas Drosophila funebris appears to display a combination of both cellular mechanisms. Furthermore, localization patterns of Par3/Bazooka suggest a self-organized, cell polarity-based origin for the variability of appendage number in S. pattersoni. Our results suggest that species deploy different combinations of apically and basally driven mechanisms to convert a two-dimensional primordium into a three-dimensional structure, and provide new directions for exploring the molecular origins of interspecies morphological variation.
Little, Shawn C, et al.Independent and coordinate trafficking of single Drosophila germ plasm mRNAs.”. Nat Cell Biol 17.5 (2015): , 17, 5, 558-68. Web.Abstract
Messenger RNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. Whereas different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single-molecule imaging, we analysed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells. We find that the germ-cell-destined transcripts nanos, cyclin B and polar granule component travel within the oocyte as ribonucleoprotein particles containing single mRNA molecules but co-assemble into multi-copy heterogeneous granules selectively at the posterior of the oocyte. The stoichiometry and dynamics of assembly indicate a defined stepwise sequence. Our data suggest that co-packaging of these transcripts ensures their effective segregation to germ cells. In contrast, compartmentalization of the germline determinant oskar mRNA into different granules limits its entry into germ cells. This exclusion is required for proper germline development.
Blythe, Shelby A, and Eric F Wieschaus. “Zygotic genome activation triggers the DNA replication checkpoint at the midblastula transition.”. Cell 160.6 (2015): , 160, 6, 1169-81. Web.Abstract
A conserved feature of the midblastula transition (MBT) is a requirement for a functional DNA replication checkpoint to coordinate cell-cycle remodeling and zygotic genome activation (ZGA). We have investigated what triggers this checkpoint during Drosophila embryogenesis. We find that the magnitude of the checkpoint scales with the quantity of transcriptionally engaged DNA. Measuring RNA polymerase II (Pol II) binding at 20 min intervals over the course of ZGA reveals that the checkpoint coincides with widespread de novo recruitment of Pol II that precedes and does not require a functional checkpoint. This recruitment drives slowing or stalling of DNA replication at transcriptionally engaged loci. Reducing Pol II recruitment in zelda mutants both reduces replication stalling and bypasses the requirement for a functional checkpoint. This suggests a model where the checkpoint functions as a feedback mechanism to remodel the cell cycle in response to nascent ZGA.
2014
He, Bing, et al.Apical constriction drives tissue-scale hydrodynamic flow to mediate cell elongation.”. Nature 508.7496 (2014): , 508, 7496, 392-6. Web.Abstract
Epithelial folding mediated by apical constriction converts flat epithelial sheets into multilayered, complex tissue structures and is used throughout development in most animals. Little is known, however, about how forces produced near the apical surface of the tissue are transmitted within individual cells to generate the global changes in cell shape that characterize tissue deformation. Here we apply particle tracking velocimetry in gastrulating Drosophila embryos to measure the movement of cytoplasm and plasma membrane during ventral furrow formation. We find that cytoplasmic redistribution during the lengthening phase of ventral furrow formation can be precisely described by viscous flows that quantitatively match the predictions of hydrodynamics. Cell membranes move with the ambient cytoplasm, with little resistance to, or driving force on, the flow. Strikingly, apical constriction produces similar flow patterns in mutant embryos that fail to form cells before gastrulation ('acellular' embryos), such that the global redistribution of cytoplasm mirrors the summed redistribution occurring in individual cells of wild-type embryos. Our results indicate that during the lengthening phase of ventral furrow formation, hydrodynamic behaviour of the cytoplasm provides the predominant mechanism transmitting apically generated forces deep into the tissue and that cell individualization is dispensable.
Polyakov, Oleg, et al.Passive mechanical forces control cell-shape change during Drosophila ventral furrow formation.”. Biophys J 107.4 (2014): , 107, 4, 998-1010. Web.Abstract
During Drosophila gastrulation, the ventral mesodermal cells constrict their apices, undergo a series of coordinated cell-shape changes to form a ventral furrow (VF) and are subsequently internalized. Although it has been well documented that apical constriction is necessary for VF formation, the mechanism by which apical constriction transmits forces throughout the bulk tissue of the cell remains poorly understood. In this work, we develop a computational vertex model to investigate the role of the passive mechanical properties of the cellular blastoderm during gastrulation. We introduce to our knowledge novel data that confirm that the volume of apically constricting cells is conserved throughout the entire course of invagination. We show that maintenance of this constant volume is sufficient to generate invagination as a passive response to apical constriction when it is combined with region-specific elasticities in the membranes surrounding individual cells. We find that the specific sequence of cell-shape changes during VF formation is critically controlled by the stiffness of the lateral and basal membrane surfaces. In particular, our model demonstrates that a transition in basal rigidity is sufficient to drive VF formation along the same sequence of cell-shape change that we observed in the actual embryo, with no active force generation required other than apical constriction.
Khan, Zia, et al.Quantitative 4D analyses of epithelial folding during Drosophila gastrulation.”. Development 141.14 (2014): , 141, 14, 2895-900. Web.Abstract
Understanding the cellular and mechanical processes that underlie the shape changes of individual cells and their collective behaviors in a tissue during dynamic and complex morphogenetic events is currently one of the major frontiers in developmental biology. The advent of high-speed time-lapse microscopy and its use in monitoring the cellular events in fluorescently labeled developing organisms demonstrate tremendous promise in establishing detailed descriptions of these events and could potentially provide a foundation for subsequent hypothesis-driven research strategies. However, obtaining quantitative measurements of dynamic shapes and behaviors of cells and tissues in a rapidly developing metazoan embryo using time-lapse 3D microscopy remains technically challenging, with the main hurdle being the shortage of robust imaging processing and analysis tools. We have developed EDGE4D, a software tool for segmenting and tracking membrane-labeled cells using multi-photon microscopy data. Our results demonstrate that EDGE4D enables quantification of the dynamics of cell shape changes, cell interfaces and neighbor relations at single-cell resolution during a complex epithelial folding event in the early Drosophila embryo. We expect this tool to be broadly useful for the analysis of epithelial cell geometries and movements in a wide variety of developmental contexts.
Di Talia, Stefano, and Eric F Wieschaus. “Simple biochemical pathways far from steady state can provide switchlike and integrated responses.”. Biophys J 107.3 (2014): , 107, 3, L1-4. Web.Abstract
Covalent modification cycles (systems in which the activity of a substrate is regulated by the action of two opposing enzymes) and ligand/receptor interactions are ubiquitous in signaling systems and their steady-state properties are well understood. However, the behavior of such systems far from steady state remains unclear. Here, we analyze the properties of covalent modification cycles and ligand/receptor interactions driven by the accumulation of the activating enzyme and the ligand, respectively. We show that for a large range of parameters both systems produce sharp switchlike response and yet allow for temporal integration of the signal, two desirable signaling properties. Ultrasensitivity is observed also in a region of parameters where the steady-state response is hyperbolic. The temporal integration properties are tunable by regulating the levels of the deactivating enzyme and receptor, as well as by adjusting the rate of accumulation of the activating enzyme and ligand. We propose that this tunability is used to generate precise responses in signaling systems.
2013
Di Talia, Stefano, et al.Posttranslational control of Cdc25 degradation terminates Drosophila's early cell-cycle program.”. Curr Biol 23.2 (2013): , 23, 2, 127-32. Web.Abstract
In most metazoans, early embryonic development is characterized by rapid mitotic divisions that are controlled by maternal mRNAs and proteins that accumulate during oogenesis. These rapid divisions pause at the midblastula transition (MBT), coinciding with a dramatic increase in gene transcription and the degradation of a subset of maternal mRNAs. In Drosophila, the cell-cycle pause is controlled by inhibitory phosphorylation of Cdk1, which in turn is driven by downregulation of the activating Cdc25 phosphatases. Here, we show that the two Drosophila Cdc25 homologs, String and Twine, differ in their dynamics and that, contrary to current models, their downregulations are not controlled by mRNA degradation but through different posttranslational mechanisms. The degradation rate of String protein gradually increases during the late syncytial cycles in a manner dependent on the nuclear-to-cytoplasmic ratio and on the DNA replication checkpoints. Twine, on the other hand, is targeted for degradation at the onset of the MBT through a switch-like mechanism controlled, like String, by the nuclear-to-cytoplasmic ratio, but not requiring the DNA replication checkpoints. We demonstrate that posttranslational control of Twine degradation ensures that the proper number of mitoses precede the MBT.
Osterfield, Miriam, et al.Three-dimensional epithelial morphogenesis in the developing Drosophila egg.”. Dev Cell 24.4 (2013): , 24, 4, 400-10. Web.Abstract
Morphogenesis of the respiratory appendages on eggshells of Drosophila species provides a powerful experimental system for studying how cell sheets give rise to complex three-dimensional structures. In Drosophila melanogaster, each of the two tubular eggshell appendages is derived from a primordium comprising two distinct cell types. Using live imaging and three-dimensional image reconstruction, we demonstrate that the transformation of this two-dimensional primordium into a tube involves out-of-plane bending followed by a sequence of spatially ordered cell intercalations. These morphological transformations correlate with the appearance of complementary distributions of myosin and Bazooka in the primordium. These distributions suggest that a two-dimensional pattern of line tensions along cell-cell edges on the apical side of the epithelium is sufficient to produce the observed changes in morphology. Computational modeling shows that this mechanism could explain the main features of tissue deformation and cell rearrangements observed during three-dimensional morphogenesis.
Wang, Yu-Chiun, Zia Khan, and Eric F Wieschaus. “Distinct Rap1 activity states control the extent of epithelial invagination via α-catenin.”. Dev Cell 25.3 (2013): , 25, 3, 299-309. Web.Abstract
Localized cell shape change initiates epithelial folding, while neighboring cell invagination determines the final depth of an epithelial fold. The mechanism that controls the extent of invagination remains unknown. During Drosophila gastrulation, a higher number of cells undergo invagination to form the deep posterior dorsal fold, whereas far fewer cells become incorporated into the initially very similar anterior dorsal fold. We find that a decrease in α-catenin activity causes the anterior fold to invaginate as extensively as the posterior fold. In contrast, constitutive activation of the small GTPase Rap1 restricts invagination of both dorsal folds in an α-catenin-dependent manner. Rap1 activity appears spatially modulated by Rapgap1, whose expression levels are high in the cells that flank the posterior fold but low in the anterior fold. We propose a model whereby distinct activity states of Rap1 modulate α-catenin-dependent coupling between junctions and actin to control the extent of epithelial invagination.
Dubuis, Julien O, et al.Positional information, in bits.”. Proc Natl Acad Sci U S A 110.41 (2013): , 110, 41, 16301-8. Web.Abstract
Cells in a developing embryo have no direct way of "measuring" their physical position. Through a variety of processes, however, the expression levels of multiple genes come to be correlated with position, and these expression levels thus form a code for "positional information." We show how to measure this information, in bits, using the gap genes in the Drosophila embryo as an example. Individual genes carry nearly two bits of information, twice as much as would be expected if the expression patterns consisted only of on/off domains separated by sharp boundaries. Taken together, four gap genes carry enough information to define a cell's location with an error bar of ~1 along the anterior/posterior axis of the embryo. This precision is nearly enough for each cell to have a unique identity, which is the maximum information the system can use, and is nearly constant along the length of the embryo. We argue that this constancy is a signature of optimality in the transmission of information from primary morphogen inputs to the output of the gap gene network.
2012
Wang, Yu-Chiun, et al.Differential positioning of adherens junctions is associated with initiation of epithelial folding.”. Nature 484.7394 (2012): , 484, 7394, 390-3. Web.Abstract
During tissue morphogenesis, simple epithelial sheets undergo folding to form complex structures. The prevailing model underlying epithelial folding involves cell shape changes driven by myosin-dependent apical constriction. Here we describe an alternative mechanism that requires differential positioning of adherens junctions controlled by modulation of epithelial apical-basal polarity. Using live embryo imaging, we show that before the initiation of dorsal transverse folds during Drosophila gastrulation, adherens junctions shift basally in the initiating cells, but maintain their original subapical positioning in the neighbouring cells. Junctional positioning in the dorsal epithelium depends on the polarity proteins Bazooka and Par-1. In particular, the basal shift that occurs in the initiating cells is associated with a progressive decrease in Par-1 levels. We show that uniform reduction of the activity of Bazooka or Par-1 results in uniform apical or lateral positioning of junctions and in each case dorsal fold initiation is abolished. In addition, an increase in the Bazooka/Par-1 ratio causes formation of ectopic dorsal folds. The basal shift of junctions not only alters the apical shape of the initiating cells, but also forces the lateral membrane of the adjacent cells to bend towards the initiating cells, thereby facilitating tissue deformation. Our data thus establish a direct link between modification of epithelial polarity and initiation of epithelial folding.
He, Bing, et al.Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster.”. Genome Res 22.12 (2012): , 22, 12, 2507-19. Web.Abstract
Heterochromatin represents a significant portion of eukaryotic genomes and has essential structural and regulatory functions. Its molecular organization is largely unknown due to difficulties in sequencing through and assembling repetitive sequences enriched in the heterochromatin. Here we developed a novel strategy using chromosomal rearrangements and embryonic phenotypes to position unmapped Drosophila melanogaster heterochromatic sequence to specific chromosomal regions. By excluding sequences that can be mapped to the assembled euchromatic arms, we identified sequences that are specific to heterochromatin and used them to design heterochromatin specific probes ("H-probes") for microarray. By comparative genomic hybridization (CGH) analyses of embryos deficient for each chromosome or chromosome arm, we were able to map most of our H-probes to specific chromosome arms. We also positioned sequences mapped to the second and X chromosomes to finer intervals by analyzing smaller deletions with breakpoints in heterochromatin. Using this approach, we were able to map >40% (13.9 Mb) of the previously unmapped heterochromatin sequences assembled by the whole-genome sequencing effort on arm U and arm Uextra to specific locations. We also identified and mapped 110 kb of novel heterochromatic sequences. Subsequent analyses revealed that sequences located within different heterochromatic regions have distinct properties, such as sequence composition, degree of repetitiveness, and level of underreplication in polytenized tissues. Surprisingly, although heterochromatin is generally considered to be transcriptionally silent, we detected region-specific temporal patterns of transcription in heterochromatin during oogenesis and early embryonic development. Our study provides a useful approach to elucidate the molecular organization and function of heterochromatin and reveals region-specific variation of heterochromatin.
Di Talia, Stefano, and Eric F Wieschaus. “Short-term integration of Cdc25 dynamics controls mitotic entry during Drosophila gastrulation.”. Dev Cell 22.4 (2012): , 22, 4, 763-74. Web.Abstract
Cells commit to mitosis by abruptly activating the mitotic cyclin-Cdk complexes. During Drosophila gastrulation, mitosis is associated with the transcriptional activation of cdc25(string), a phosphatase that activates Cdk1. Here, we demonstrate that the switch-like entry into mitosis observed in the Drosophila embryo during the 14(th) mitotic cycle is timed by the dynamics of Cdc25(string) accumulation. The switch operates as a short-term integrator, a property that can improve the reliable control of timing of mitosis. The switch is independent of the positive feedback between Cdk1 and Cdc25(string) and of the double negative feedback between Cdk1 and Wee1. We propose that the properties of the mitotic switch are established by the out-of-equilibrium properties of the covalent modification cycle controlling Cdk1 activity. Such covalent modification cycles, triggered by transcriptional expression of the activating enzymes, might be a widespread strategy to obtain reliable and switch-like control of cell decisions.
Grimm, Oliver, et al.Torso RTK controls Capicua degradation by changing its subcellular localization.”. Development 139.21 (2012): , 139, 21, 3962-8. Web.Abstract
The transcriptional repressor Capicua (Cic) controls multiple aspects of Drosophila embryogenesis and has been implicated in vertebrate development and human diseases. Receptor tyrosine kinases (RTKs) can antagonize Cic-dependent gene repression, but the mechanisms responsible for this effect are not fully understood. Based on genetic and imaging studies in the early Drosophila embryo, we found that Torso RTK signaling can increase the rate of Cic degradation by changing its subcellular localization. We propose that Cic is degraded predominantly in the cytoplasm and show that Torso reduces the stability of Cic by controlling the rates of its nucleocytoplasmic transport. This model accounts for the experimentally observed spatiotemporal dynamics of Cic in the early embryo and might explain RTK-dependent control of Cic in other developmental contexts.
Gelbart, Michael A, et al.Volume conservation principle involved in cell lengthening and nucleus movement during tissue morphogenesis.”. Proc Natl Acad Sci U S A 109.47 (2012): , 109, 47, 19298-303. Web.Abstract
Tissue morphogenesis is the process in which coordinated movements and shape changes of large numbers of cells form tissues, organs, and the internal body structure. Understanding morphogenetic movements requires precise measurements of whole-cell shape changes over time. Tissue folding and invagination are thought to be facilitated by apical constriction, but the mechanism by which changes near the apical cell surface affect changes along the entire apical-basal axis of the cell remains elusive. Here, we developed Embryo Development Geometry Explorer, an approach for quantifying rapid whole-cell shape changes over time, and we combined it with deep-tissue time-lapse imaging based on fast two-photon microscopy to study Drosophila ventral furrow formation. We found that both the cell lengthening along the apical-basal axis and the movement of the nucleus to the basal side proceeded stepwise and were correlated with apical constriction. Moreover, cell volume lost apically due to constriction largely balanced the volume gained basally by cell lengthening. The volume above the nucleus was conserved during its basal movement. Both apical volume loss and cell lengthening were absent in mutants showing deficits in the contractile cytoskeleton underlying apical constriction. We conclude that a single mechanical mechanism involving volume conservation and apical constriction-induced basal movement of cytoplasm accounts quantitatively for the cell shape changes and the nucleus movement in Drosophila ventral furrow formation. Our study provides a comprehensive quantitative analysis of the fast dynamics of whole-cell shape changes during tissue folding and points to a simplified model for Drosophila gastrulation.
2011
Little, Shawn C, et al.The formation of the Bicoid morphogen gradient requires protein movement from anteriorly localized mRNA.”. PLoS Biol 93 (2011): , 9, 3, e1000596. Web.Abstract
The Bicoid morphogen gradient directs the patterning of cell fates along the anterior-posterior axis of the syncytial Drosophila embryo and serves as a paradigm of morphogen-mediated patterning. The simplest models of gradient formation rely on constant protein synthesis and diffusion from anteriorly localized source mRNA, coupled with uniform protein degradation. However, currently such models cannot account for all known gradient characteristics. Recent work has proposed that bicoid mRNA spatial distribution is sufficient to produce the observed protein gradient, minimizing the role of protein transport. Here, we adapt a novel method of fluorescent in situ hybridization to quantify the global spatio-temporal dynamics of bicoid mRNA particles. We determine that >90% of all bicoid mRNA is continuously present within the anterior 20% of the embryo. bicoid mRNA distribution along the body axis remains nearly unchanged despite dynamic mRNA translocation from the embryo core to the cortex. To evaluate the impact of mRNA distribution on protein gradient dynamics, we provide detailed quantitative measurements of nuclear Bicoid levels during the formation of the protein gradient. We find that gradient establishment begins 45 minutes after fertilization and that the gradient requires about 50 minutes to reach peak levels. In numerical simulations of gradient formation, we find that incorporating the actual bicoid mRNA distribution yields a closer prediction of the observed protein dynamics compared to modeling protein production from a point source at the anterior pole. We conclude that the spatial distribution of bicoid mRNA contributes to, but cannot account for, protein gradient formation, and therefore that protein movement, either active or passive, is required for gradient formation.
Drocco, Jeffrey A, et al.Measurement and perturbation of morphogen lifetime: effects on gradient shape.”. Biophys J 101.8 (2011): , 101, 8, 1807-15. Web.Abstract
Protein lifetime is of critical importance for most biological processes and plays a central role in cell signaling and embryonic development, where it impacts the absolute concentration of signaling molecules and, potentially, the shape of morphogen gradients. Early conceptual and mathematical models of gradient formation proposed that steady-state gradients are established by an equilibration between the lifetime of a morphogen and its rates of synthesis and diffusion, though whether gradients in fact reach steady state before being read out is a matter of controversy. In any case, this class of models predicts that protein lifetime is a key determinant of both the time to steady state and the spatial extent of a gradient. Using a method that employs repeated photoswitching of a fusion of the morphogen Bicoid (Bcd) and the photoconvertible fluorescent protein Dronpa, we measure and modify the lifetime of Dronpa-Bcd in living Drosophila embryos. We find that the lifetime of Bcd is dynamic, changing from 50 min before mitotic cycle 14 to 15 min during cellularization. Moreover, by measuring total quantities of Bcd over time, we find that the gradient does not reach steady state. Finally, using a nearly continuous low-level conversion to the dark state of Dronpa-Bcd to mimic the effect of increased degradation, we demonstrate that perturbation of protein lifetime changes the characteristic length of the gradient, providing direct support for a mechanism based on synthesis, diffusion, and degradation.
Little, Shawn C, and Eric F Wieschaus. “Shifting patterns: merging molecules, morphogens, motility, and methodology.”. Dev Cell 21.1 (2011): , 21, 1, 2-4. Web.Abstract
We highlight crucial technological progress of the past ten years that permits quantitative analysis of cellular behavior. Adapting these methods to the study of embryogenesis will be essential to advance our understanding of development in the coming decade.
2010
Grimm, Oliver, and Eric Wieschaus. “The Bicoid gradient is shaped independently of nuclei.”. Development 137.17 (2010): , 137, 17, 2857-62. Web.Abstract
Morphogen gradients provide embryos with positional information, yet how they form is not understood. Binding of the morphogen to receptors could affect the formation of the morphogen gradient, in particular if the number of morphogen binding sites changes with time. For morphogens that function as transcription factors, the final distribution can be heavily influenced by the number of nuclear binding sites. Here, we have addressed the role of the increasing number of nuclei during the formation of the Bicoid gradient in embryos of Drosophila melanogaster. Deletion of a short stretch of sequence in Bicoid impairs its nuclear accumulation. This effect is due to a approximately 4-fold decrease in nuclear import rate and a approximately 2-fold reduction in nuclear residence time compared with the wild-type protein. Surprisingly, the shape of the resulting anterior-posterior gradient as well as the centre-surface distribution are indistinguishable from those of the normal gradient. This suggests that nuclei do not shape the Bicoid gradient but instead function solely during its interpretation.
Martin, Adam C, et al.Integration of contractile forces during tissue invagination.”. J Cell Biol 188.5 (2010): , 188, 5, 735-49. Web.Abstract
Contractile forces generated by the actomyosin cytoskeleton within individual cells collectively generate tissue-level force during epithelial morphogenesis. During Drosophila mesoderm invagination, pulsed actomyosin meshwork contractions and a ratchet-like stabilization of cell shape drive apical constriction. Here, we investigate how contractile forces are integrated across the tissue. Reducing adherens junction (AJ) levels or ablating actomyosin meshworks causes tissue-wide epithelial tears, which release tension that is predominantly oriented along the anterior-posterior (a-p) embryonic axis. Epithelial tears allow cells normally elongated along the a-p axis to constrict isotropically, which suggests that apical constriction generates anisotropic epithelial tension that feeds back to control cell shape. Epithelial tension requires the transcription factor Twist, which stabilizes apical myosin II, promoting the formation of a supracellular actomyosin meshwork in which radial actomyosin fibers are joined end-to-end at spot AJs. Thus, pulsed actomyosin contractions require a supracellular, tensile meshwork to transmit cellular forces to the tissue level during morphogenesis.
Grimm, Oliver, Mathieu Coppey, and Eric Wieschaus. “Modelling the Bicoid gradient.”. Development 137.14 (2010): , 137, 14, 2253-64. Web.Abstract
Morphogen gradients provide embryonic tissues with positional information by inducing target genes at different concentration thresholds and thus at different positions. The Bicoid morphogen gradient in Drosophila melanogaster embryos has recently been analysed quantitatively, yet how it forms remains a matter of controversy. Several biophysical models that rely on production, diffusion and degradation have been formulated to account for the observed dynamics of the Bicoid gradient, but no one model can account for all its characteristics. Here, we discuss how existing data on this gradient fit the various proposed models and what aspects of gradient formation these models fail to explain. We suggest that knowing a few additional parameters, such as the lifetime of Bicoid, would help to identify and develop better models of Bicoid gradient formation.
2009
Lu, Xuemin, et al.Coupling of zygotic transcription to mitotic control at the Drosophila mid-blastula transition.”. Development 136.12 (2009): , 136, 12, 2101-10. Web.Abstract
One of the most prominent features at the mid-blastula transition (MBT) observed in most embryos is a pause in cell cycle regulated by the nucleocytoplasmic (N/C) ratio. By using chromosome rearrangements to manipulate the DNA content of embryos, we determined that the threshold for this cell cycle pause in Drosophila is about 70% of the DNA content normally present at cycle 14. Embryos with DNA contents around this value show intermediate cell cycle behaviors. Some pause at cycle 14, some at cycle 15, and some form patches arrested in different mitotic cycles. A second feature at MBT is a massive increase in zygotic transcription and a parallel degradation of maternally supplied RNAs. To determine whether these changes in gene expression are governed by the same N/C ratio that controls cell cycle pause, we compared gene expression in haploid and diploid Drosophila embryos. We find that most maternal RNA degradation and most new transcription correlate with absolute time or developmental stage, and are timed independently of the N/C ratio. We identify a class of zygotically active genes whose expression depends on the N/C ratio and which are only expressed at cycle 15 in haploids. In embryos with patchy cell cycle behavior due to threshold DNA contents, the expression of these genes correlates tightly with the boundaries of the mitotic patches, suggesting either that the mechanism that pauses the mitotic cycle is the same as the one that measures the N/C ratio, or that it is tightly coupled to the mechanism controlling zygotic transcription of N/C ratio genes at the MBT.
Martin, Adam C, Matthias Kaschube, and Eric F Wieschaus. “Pulsed contractions of an actin-myosin network drive apical constriction.”. Nature 457.7228 (2009): , 457, 7228, 495-9. Web.Abstract
Apical constriction facilitates epithelial sheet bending and invagination during morphogenesis. Apical constriction is conventionally thought to be driven by the continuous purse-string-like contraction of a circumferential actin and non-muscle myosin-II (myosin) belt underlying adherens junctions. However, it is unclear whether other force-generating mechanisms can drive this process. Here we show, with the use of real-time imaging and quantitative image analysis of Drosophila gastrulation, that the apical constriction of ventral furrow cells is pulsed. Repeated constrictions, which are asynchronous between neighbouring cells, are interrupted by pauses in which the constricted state of the cell apex is maintained. In contrast to the purse-string model, constriction pulses are powered by actin-myosin network contractions that occur at the medial apical cortex and pull discrete adherens junction sites inwards. The transcription factors Twist and Snail differentially regulate pulsed constriction. Expression of snail initiates actin-myosin network contractions, whereas expression of twist stabilizes the constricted state of the cell apex. Our results suggest a new model for apical constriction in which a cortical actin-myosin cytoskeleton functions as a developmentally controlled subcellular ratchet to reduce apical area incrementally.
2008
Sokac, Anna Marie, and Eric Wieschaus. “Local actin-dependent endocytosis is zygotically controlled to initiate Drosophila cellularization.”. Dev Cell 14.5 (2008): , 14, 5, 775-86. Web.Abstract
In early Drosophila embryos, several mitotic cycles proceed with aborted cytokinesis before a modified cytokinesis, called cellularization, finally divides the syncytium into individual cells. Here, we find that scission of endocytic vesicles from the plasma membrane (PM) provides a control point to regulate the furrowing events that accompany this development. At early mitotic cycles, local furrow-associated endocytosis is controlled by cell cycle progression, whereas at cellularization, which occurs in a prolonged interphase, it is controlled by expression of the zygotic gene nullo. nullo mutations impair cortical F-actin accumulation and scission of endocytic vesicles, such that membrane tubules remain tethered to the PM and deplete structural components from the furrows, precipitating furrow regression. Thus, Nullo regulates scission to restrain endocytosis of proteins essential for furrow stabilization at the onset of cellularization. We propose that developmentally regulated endocytosis can coordinate actin/PM remodeling to directly drive furrow dynamics during morphogenesis.
Gregor, Thomas, Alistair P McGregor, and Eric F Wieschaus. “Shape and function of the Bicoid morphogen gradient in dipteran species with different sized embryos.”. Dev Biol 316.2 (2008): , 316, 2, 350-8. Web.Abstract
The Bicoid morphogen evolved approximately 150 MYA from a Hox3 duplication and is only found in higher dipterans. A major difference between dipteran species, however, is the size of the embryo, which varies up to 5-fold. Although the expression of developmental factors scale with egg length, it remains unknown how this scaling is achieved. To test whether scaling is accounted for by the properties of Bicoid, we expressed eGFP fused to the coding region of bicoid from three dipteran species in transgenic Drosophila embryos using the Drosophila bicoid cis-regulatory and mRNA localization sequences. In such embryos, we find that Lucilia sericata and Calliphora vicina Bicoid produce gradients very similar to the endogenous Drosophila gradient and much shorter than what they would have produced in their own respective species. The common shape of the Drosophila, Lucilia and Calliphora Bicoid gradients appears to be a conserved feature of the Bicoid protein. Surprisingly, despite their similar distributions, we find that Bicoid from Lucilia and Calliphora do not rescue Drosophila bicoid mutants, suggesting that that Bicoid proteins have evolved species-specific functional amino acid differences. We also found that maternal expression and anteriorly localization of proteins other than Bcd does not necessarily give rise to a gradient; eGFP produced a uniform protein distribution. However, a shallow gradient was observed using eGFP-NLS, suggesting nuclear localization may be necessary but not sufficient for gradient formation.
Sokac, Anna Marie, and Eric Wieschaus. “Zygotically controlled F-actin establishes cortical compartments to stabilize furrows during Drosophila cellularization.”. J Cell Sci 121.11 (2008): , 121, 11, 1815-24. Web.Abstract
Cortical compartments partition proteins and membrane at the cell surface to define regions of specialized function. Here we ask how cortical compartments arise along the plasma membrane furrows that cellularize the early Drosophila embryo, and investigate the influence that this compartmentalization has on furrow ingression. We find that the zygotic gene product Nullo aids the establishment of discrete cortical compartments, called furrow canals, which form at the tip of incipient furrows. Upon nullo loss-of-function, proteins that are normally restricted to adjacent lateral regions of the furrow, such as Neurotactin and Discs large, spread into the furrow canals. At the same time, cortical components that should concentrate in furrow canals, such as Myosin 2 (Zipper) and Anillin (Scraps), are missing from some furrows. Depletion of these cortical components from the furrow canal compartments precipitates furrow regression. Contrary to previous models, we find that furrow compartmentalization does not require cell-cell junctions that border the furrow canals. Instead, compartmentalization is disrupted by treatments that reduce levels of cortical F-actin. Because the earliest uniform phenotype detected in nullo mutants is reduced levels of F-actin at furrow canals, we propose that Nullo compartmentalizes furrows via its regulation of F-actin, thus stabilizing furrows and insuring their ingression to complete cellularization.
2007
Goodliffe, Julie M, Michael D Cole, and Eric Wieschaus. “Coordinated regulation of Myc trans-activation targets by Polycomb and the Trithorax group protein Ash1.”. BMC Mol Biol 8 (2007): , 8, 40. Web.Abstract
BACKGROUND: The Myc oncoprotein is a transcriptional regulator whose function is essential for normal development. Myc is capable of binding to 10% of the mammalian genome, and it is unclear how a developing embryo controls the DNA binding of its abundant Myc proteins in order to avoid Myc's potential for inducing tumorigenesis. RESULTS: To identify chromatin binding proteins with a potential role in controlling Myc activity, we established a genetic assay for dMyc activity in Drosophila. We conducted a genome-wide screen using this assay, and identified the Trithorax Group protein Ash1 as a modifier of dMyc activity. Ash1 is a histone methyltransferase known for its role in opposing repression by Polycomb. Using RNAi in the embryo and Affymetrix microarrays, we show that ash1 RNAi causes the increased expression of many genes, suggesting that it is directly or indirectly required for repression in the embryo, in contrast to its known role in maintenance of activation. Many of these genes also respond similarly upon depletion of Pc and pho transcripts, as determined by concurrent microarray analysis of Pc and pho RNAi embryos, suggesting that the three are required for low levels of expression of a common set of targets. Further, many of these overlapping targets are also activated by Myc overexpression. We identify a second group of genes whose expression in the embryo requires Ash1, consistent with its previously established role in maintenance of activation. We find that this second group of Ash1 targets overlaps those activated by Myc and that ectopic Myc overcomes their requirement for Ash1. CONCLUSION: Genetic, genomic and chromatin immunoprecipitation data suggest a model in which Pc, Ash1 and Pho are required to maintain a low level of expression of embryonic targets of activation by Myc, and that this occurs, directly or indirectly, by a combination of disparate chromatin modifications.
De Renzis, Stefano, et al.Unmasking activation of the zygotic genome using chromosomal deletions in the Drosophila embryo.”. PLoS Biol 55 (2007): , 5, 5, e117. Web.Abstract
During the maternal-to-zygotic transition, a developing embryo integrates post-transcriptional regulation of maternal mRNAs with transcriptional activation of its own genome. By combining chromosomal ablation in Drosophila with microarray analysis, we characterized the basis of this integration. We show that the expression profile for at least one third of zygotically active genes is coupled to the concomitant degradation of the corresponding maternal mRNAs. The embryo uses transcription and degradation to generate localized patterns of expression, and zygotic transcription to degrade distinct classes of maternal transcripts. Although degradation does not appear to involve a simple regulatory code, the activation of the zygotic genome starts from intronless genes sharing a common cis-element. This cis-element interacts with a single protein, the Bicoid stability factor, and acts as a potent enhancer capable of timing the activity of an exogenous transactivator. We propose that this regulatory mode links morphogen gradients with temporal regulation during the maternal-to-zygotic transition.
Gregor, Thomas, et al.Probing the limits to positional information.”. Cell 130.1 (2007): , 130, 1, 153-64. Web.Abstract
The reproducibility and precision of biological patterning is limited by the accuracy with which concentration profiles of morphogen molecules can be established and read out by their targets. We consider four measures of precision for the Bicoid morphogen in the Drosophila embryo: the concentration differences that distinguish neighboring cells, the limits set by the random arrival of Bicoid molecules at their targets (which depends on absolute concentration), the noise in readout of Bicoid by the activation of Hunchback, and the reproducibility of Bicoid concentration at corresponding positions in multiple embryos. We show, through a combination of different experiments, that all of these quantities are approximately 10%. This agreement among different measures of accuracy indicates that the embryo is not faced with noisy input signals and readout mechanisms; rather, the system exerts precise control over absolute concentrations and responds reliably to small concentration differences, approaching the limits set by basic physical principles.
Gregor, Thomas, et al.Stability and nuclear dynamics of the bicoid morphogen gradient.”. Cell 130.1 (2007): , 130, 1, 141-52. Web.Abstract
Patterning in multicellular organisms results from spatial gradients in morphogen concentration, but the dynamics of these gradients remain largely unexplored. We characterize, through in vivo optical imaging, the development and stability of the Bicoid morphogen gradient in Drosophila embryos that express a Bicoid-eGFP fusion protein. The gradient is established rapidly (approximately 1 hr after fertilization), with nuclear Bicoid concentration rising and falling during mitosis. Interphase levels result from a rapid equilibrium between Bicoid uptake and removal. Initial interphase concentration in nuclei in successive cycles is constant (+/-10%), demonstrating a form of gradient stability, but it subsequently decays by approximately 30%. Both direct photobleaching measurements and indirect estimates of Bicoid-eGFP diffusion constants (D < or = 1 microm(2)/s) provide a consistent picture of Bicoid transport on short ( approximately min) time scales but challenge traditional models of long-range gradient formation. A new model is presented emphasizing the possible role of nuclear dynamics in shaping and scaling the gradient.
2006
De Renzis, Stefano, et al.Dorsal-ventral pattern of Delta trafficking is established by a Snail-Tom-Neuralized pathway.”. Dev Cell 10.2 (2006): , 10, 2, 257-64. Web.Abstract
The intracellular trafficking of the Notch ligand Delta plays an important role in the activation of the Notch pathway. We have addressed the snail-dependent regulation of Delta trafficking during the plasma membrane growth of the mesoderm in the Drosophila embryo. We show that Delta is retained in endocytic vesicles in the mesoderm but expressed on the surface of the adjacent ectoderm. This trafficking pattern requires Neuralized. We developed a protocol based on chromosomal deletion and microarray analysis that led to the identification of tom as the target of snail regulating Delta trafficking. Snail represses Tom expression in the mesoderm and thereby activates Delta trafficking. Overexpression of Tom abolishes Delta trafficking and signaling to the adjacent mesoectoderm. Loss of Tom produces mesoderm-type Delta trafficking in the entire blastoderm epithelium and an expansion of mesoectoderm gene expression. We propose that Tom antagonizes the activity of Neuralized and thus establishes a sharp mesoderm-mesoectoderm boundary of Notch signaling.
Frydman, Horacio M, et al.Somatic stem cell niche tropism in Wolbachia.”. Nature 441.7092 (2006): , 441, 7092, 509-12. Web.Abstract
Wolbachia are intracellular bacteria found in the reproductive tissue of all major groups of arthropods. They are transmitted vertically from the female hosts to their offspring, in a pattern analogous to mitochondria inheritance. But Wolbachia phylogeny does not parallel that of the host, indicating that horizontal infectious transmission must also occur. Insect parasitoids are considered the most likely vectors, but the mechanism for horizontal transfer is largely unknown. Here we show that newly introduced Wolbachia cross several tissues and infect the germline of the adult Drosophila melanogaster female. Through investigation of bacterial migration patterns during the course of infection, we found that Wolbachia reach the germline through the somatic stem cell niche in the D. melanogaster germarium. In addition, our data suggest that Wolbachia are highly abundant in the somatic stem cell niche of long-term infected hosts, implying that this location may also contribute to efficient vertical transmission. This is, to our knowledge, the first report of an intracellular parasite displaying tropism for a stem cell niche.
2005
Gregor, Thomas, et al.Diffusion and scaling during early embryonic pattern formation.”. Proc Natl Acad Sci U S A 102.51 (2005): , 102, 51, 18403-7. Web.Abstract
Development of spatial patterns in multicellular organisms depends on gradients in the concentration of signaling molecules that control gene expression. In the Drosophila embryo, Bicoid (Bcd) morphogen controls cell fate along 70% of the anteroposterior axis but is translated from mRNA localized at the anterior pole. Gradients of Bcd and other morphogens are thought to arise through diffusion, but this basic assumption has never been rigorously tested in living embryos. Furthermore, because diffusion sets a relationship between length and time scales, it is hard to see how patterns of gene expression established by diffusion would scale proportionately as egg size changes during evolution. Here, we show that the motion of inert molecules through the embryo is well described by the diffusion equation on the relevant length and time scales, and that effective diffusion constants are essentially the same in closely related dipteran species with embryos of very different size. Nonetheless, patterns of gene expression in these different species scale with egg length. We show that this scaling can be traced back to scaling of the Bcd gradient itself. Our results, together with constraints imposed by the time scales of development, suggest that the mechanism for scaling is a species-specific adaptation of the Bcd lifetime.
Dawes-Hoang, Rachel E, et al.folded gastrulation, cell shape change and the control of myosin localization.”. Development 132.18 (2005): , 132, 18, 4165-78. Web.Abstract
The global cell movements that shape an embryo are driven by intricate changes to the cytoarchitecture of individual cells. In a developing embryo, these changes are controlled by patterning genes that confer cell identity. However, little is known about how patterning genes influence cytoarchitecture to drive changes in cell shape. In this paper, we analyze the function of the folded gastrulation gene (fog), a known target of the patterning gene twist. Our analysis of fog function therefore illuminates a molecular pathway spanning all the way from patterning gene to physical change in cell shape. We show that secretion of Fog protein is apically polarized, making this the earliest polarized component of a pathway that ultimately drives myosin to the apical side of the cell. We demonstrate that fog is both necessary and sufficient to drive apical myosin localization through a mechanism involving activation of myosin contractility with actin. We determine that this contractility driven form of localization involves RhoGEF2 and the downstream effector Rho kinase. This distinguishes apical myosin localization from basal myosin localization, which we find not to require actinomyosin contractility or FOG/RhoGEF2/Rho-kinase signaling. Furthermore, we demonstrate that once localized apically, myosin continues to contract. The force generated by continued myosin contraction is translated into a flattening and constriction of the cell surface through a tethering of the actinomyosin cytoskeleton to the apical adherens junctions. Our analysis of fog function therefore provides a direct link from patterning to cell shape change.
Goodliffe, Julie M, Eric Wieschaus, and Michael D Cole. “Polycomb mediates Myc autorepression and its transcriptional control of many loci in Drosophila.”. Genes Dev 19.24 (2005): , 19, 24, 2941-6. Web.Abstract
Aberrant accumulation of the Myc oncoprotein propels proliferation and induces carcinogenesis. In normal cells, however, an abundance of Myc protein represses transcription at the c-myc locus. Cancer cells often lose this autorepression. We examined the control of myc in Drosophila and show here that the Drosophila ortholog, dmyc, also undergoes autorepression. We find that the developmental repressor Polycomb (Pc) is required for dmyc autorepression, and that this Pc-dMyc-mediated repression spreads across an 875-kb region encompassing the dmyc gene. To further investigate the relationship between Myc and Polycomb, we used microarrays to identify genes regulated by each, and identify a striking relationship between the two: A large set of dMyc activation targets is normally repressed by Pc, and 73% of dMyc repression targets require Pc for this repression. Chromatin immunoprecipitation confirmed that many dMyc-Pc-repressed loci have an epigenetic mark recognized by Pc. Our results suggest a novel relationship between Myc and Polycomb, wherein Myc enhances Polycomb repression in order to repress targets, and Myc suppresses Polycomb repression in order to activate targets.
2004
Thomas, Jeffrey H, and Eric Wieschaus. “src64 and tec29 are required for microfilament contraction during Drosophila cellularization.”. Development 131.4 (2004): , 131, 4, 863-71. Web.Abstract
Formation of the Drosophila cellular blastoderm involves both membrane invagination and cytoskeletal regulation. Mutations in src64 and tec29 reveal a novel role for these genes in controlling contraction of the actin-myosin microfilament ring during this process. Although membrane invagination still proceeds in mutant embryos, its depth is not uniform, and basal closure of the cells does not occur during late cellularization. Double-mutant analysis between scraps, a mutation in anillin that eliminates microfilament rings, and bottleneck suggests that microfilaments can still contract even though they are not organized into rings. However, the failure of rings to contract in the src64 bottleneck double mutant suggests that src64 is required for microfilament ring contraction even in the absence of Bottleneck protein. Our results suggest that src64-dependent microfilament ring contraction is resisted by Bottleneck to create tension and coordinate membrane invagination during early cellularization. The absence of Bottleneck during late cellularization allows src64-dependent microfilament ring constriction to drive basal closure.
Tolwinski, Nicholas S, and Eric Wieschaus. “A nuclear function for armadillo/beta-catenin.”. PLoS Biol 24 (2004): , 2, 4, E95. Web.Abstract
The Wnt signaling pathway provides key information during development of vertebrates and invertebrates, and mutations in this pathway lead to various forms of cancer. Wnt binding to its receptor causes the stabilization and nuclear localization of beta-catenin. Nuclear beta-catenin then functions to activate transcription in conjunction with the transcription factor TCF. A recent report has challenged this basic precept of the Wnt signaling field, arguing that the nuclear localization of beta-catenin may be unrelated to its function and that beta-catenin functions at the plasma membrane to activate this signaling pathway. Here we present evidence that the pathway in fact does depend on the nuclear localization of beta-catenin. We reexamine the functionality of various truncations of beta-catenin and find that only the most severe truncations are true signaling-null mutations. Further, we define a signaling-null condition and use it to show that membrane-tethered beta-catenin is insufficient to activate transcription. We also define two novel loss-of-function mutations that are not truncations, but are missense point mutations that retain protein stability. These alleles allow us to show that the membrane-bound form of activated beta-catenin does indeed depend on the endogenous protein. Further, this activity is dependent on the presence of the C-terminus-specific negative regulator Chibby. Our data clearly show that nuclear localization of beta-catenin is in fact necessary for Wnt pathway activation.
Zallen, Jennifer A, and Eric Wieschaus. “Patterned gene expression directs bipolar planar polarity in Drosophila.”. Dev Cell 63 (2004): , 6, 3, 343-55. Print.Abstract
During convergent extension in Drosophila, polarized cell movements cause the germband to narrow along the dorsal-ventral (D-V) axis and more than double in length along the anterior-posterior (A-P) axis. This tissue remodeling requires the correct patterning of gene expression along the A-P axis, perpendicular to the direction of cell movement. Here, we demonstrate that A-P patterning information results in the polarized localization of cortical proteins in intercalating cells. In particular, cell fate differences conferred by striped expression of the even-skipped and runt pair-rule genes are both necessary and sufficient to orient planar polarity. This polarity consists of an enrichment of nonmuscle myosin II at A-P cell borders and Bazooka/PAR-3 protein at the reciprocal D-V cell borders. Moreover, bazooka mutants are defective for germband extension. These results indicate that spatial patterns of gene expression coordinate planar polarity across a multicellular population through the localized distribution of proteins required for cell movement.
Tolwinski, Nicholas S, and Eric Wieschaus. “Rethinking WNT signaling.”. Trends Genet 20.4 (2004): , 20, 4, 177-81. Web.Abstract
Recent research on the WNT signaling pathway warrants a reassessment of the basic mechanism that transmits signal from the membrane-bound receptor to the nucleus. This article incorporates these findings into a revised model for pathway activation. We propose that the control of Axin stability, rather than the control of ZW3 phosphorylation of the Armadillo protein, is the key step in signaling. Axin degradation is controlled by a stabilizing effect of ZW3-dependent phosphorylation, and a destabilizing effect of active Arrow. Removing Axin enables Armadillo to accumulate and re-localize to the nucleus. We argue that nuclear localization of Armadillo is required for transcriptional pathway activity. Finally, we speculate on the effects this revision will have on the major questions facing the WNT field of research.
2003
Dawes-Hoang, Rachel E, Jennifer A Zallen, and Eric F Wieschaus. “Bringing classical embryology to C elegans gastrulation.”. Dev Cell 41 (2003): , 4, 1, 6-8. Print.Abstract
In a recent paper, Lee and Goldstein develop an explant assay that recapitulates key aspects of gastrulation in C elegans and permits classical embryological manipulations. The resulting detailed analysis of cell behavior will ultimately extend to broader issues, such as, whether morphogenesis can be described as the sum of single-cell events or if unique phenomena emerge at the multicellular level.
Tolwinski, Nicholas S, et al.Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity.”. Dev Cell 43 (2003): , 4, 3, 407-18. Print.Abstract
Activation of the Wnt signaling cascade provides key signals during development and in disease. Here we provide evidence, by designing a Wnt receptor with ligand-independent signaling activity, that physical proximity of Arrow (LRP) to the Wnt receptor Frizzled-2 triggers the intracellular signaling cascade. We have uncovered a branch of the Wnt pathway in which Armadillo activity is regulated concomitantly with the levels of Axin protein. The intracellular pathway bypasses Gsk3beta/Zw3, the kinase normally required for controlling beta-catenin/Armadillo levels, suggesting that modulated degradation of Armadillo is not required for Wnt signaling. We propose that Arrow (LRP) recruits Axin to the membrane, and that this interaction leads to Axin degradation. As a consequence, Armadillo is no longer bound by Axin, resulting in nuclear signaling by Armadillo.
2002
Hunter, Christine, et al.Conserved domains of the Nullo protein required for cell-surface localization and formation of adherens junctions.”. Mol Biol Cell 13.1 (2002): , 13, 1, 146-57. Web.Abstract
During cellularization, the Drosophila melanogaster embryo undergoes a transition from syncytial to cellular blastoderm with the de novo generation of a polarized epithelial sheet in the cortex of the embryo. This process couples cytokinesis with the establishment of apical, basal, and lateral membrane domains that are separated by two spatially distinct adherens-type junctions. In nullo mutant embryos, basal junctions fail to form at the onset of cellularization, leading to the failure of cleavage furrow invagination and the generation of multinucleate cells. Nullo is a novel protein that appears to stabilize the initial accumulation of cadherins and catenins as they form a mature basal junction. In this article we characterize a nullo homologue from D. virilis and identify conserved domains of Nullo that are required for basal junction formation. We also demonstrate that Nullo is a myristoylprotein and that the myristate group acts in conjunction with a cluster of basic amino acids to target Nullo to the plasma membrane. The membrane association of Nullo is required in vivo for its role in basal junction formation and for its ability to block apical junction formation when ectopically expressed during late cellularization.
Ahmed, Yashi, Ali Nouri, and Eric Wieschaus. “Drosophila Apc1 and Apc2 regulate Wingless transduction throughout development.”. Development 129.7 (2002): , 129, 7, 1751-62. Print.Abstract
Inactivation of the Adenomatous Polyposis Coli (APC) tumor suppressor triggers the development of most colorectal carcinomas. APC is required for targeted degradation of beta-catenin, the central transcriptional activator in the Wnt/Wingless (Wg) signal transduction pathway; however, the precise biochemical functions of APC remain uncertain. The two Drosophila homologs of APC (Apc1 and Apc2) appear to have predominantly different tissue distributions, different subcellular localizations and mutually exclusive phenotypes upon inactivation. Unexpectedly, we have found that despite these differences, simultaneous reduction in both Drosophila Apc proteins results in the global nuclear accumulation of beta-catenin and the constitutive activation of Wg transduction throughout development. This redundancy extends even to functions previously thought to be specific to the individual Apc homologs. Together, these results reveal that the combined activity of Apc1 and Apc2 allows a tight regulation of transcriptional activation by beta-catenin and suggest that APC proteins are required for the regulation of Wnt transduction in all cells.
Houchmandzadeh, Bahram, Eric Wieschaus, and Stanislas Leibler. “Establishment of developmental precision and proportions in the early Drosophila embryo.”. Nature 415.6873 (2002): , 415, 6873, 798-802. Web.Abstract
During embryonic development, orderly patterns of gene expression eventually assign each cell in the embryo its particular fate. For the anteroposterior axis of the Drosophila embryo, the first step in this process depends on a spatial gradient of the maternal morphogen Bicoid (Bcd). Positional information of this gradient is transmitted to downstream gap genes, each occupying a well defined spatial domain. We determined the precision of the initial process by comparing expression domains in different embryos. Here we show that the Bcd gradient displays a high embryo-to-embryo variability, but that this noise in the positional information is strongly decreased ('filtered') at the level of hunchback (hb) gene expression. In contrast to the Bcd gradient, the hb expression pattern already includes the information about the scale of the embryo. We show that genes known to interact directly with Hb are not responsible for its spatial precision, but that the maternal gene staufen may be crucial.
Lecuit, Thomas, and Eric Wieschaus. “Junctions as organizing centers in epithelial cells? A fly perspective.”. Traffic 32 (2002): , 3, 2, 92-7. Print.Abstract
Junctions isolate the apical from the basal-lateral surfaces and are required for the polarization of epithelial cells. Genetics of junction formation in Drosophila has identified numerous transmembrane and scaffolding proteins that assemble in membrane-associated complexes. This review focuses on recent findings suggesting that junctions are autonomous organizing centers for the establishment and maintenance of apical-basal polarity in epithelial cells as well as other polarized processes such as the orientation of the division apparatus.
Lecuit, Thomas, Reba Samanta, and Eric Wieschaus. “slam encodes a developmental regulator of polarized membrane growth during cleavage of the Drosophila embryo.”. Dev Cell 24 (2002): , 2, 4, 425-36. Print.Abstract
Cellularization of the Drosophila embryo is a specialized form of cytokinesis that couples membrane growth with the formation of a polarized epithelium. We have identified a gene essential for polarized growth of the plasma membrane during cellularization. In slam mutant embryos, the furrow canal is disorganized, and polarized insertion of transmembrane proteins is disrupted. slam shows a striking developmental induction during the slow phase of cellularization, and Slam protein localizes to the furrow canal and the basal junction. Slam colocalizes with the junctional proteins Arm/beta-catenin, the PDZ domain-containing protein Dlt, and Myosin and is also required for their proper membrane localization. Our results suggest that developmental induction of Slam organizes the polarized growth of membrane via the recruitment of membrane-targeting proteins at adherens junctions.
1986
Gergen, Peter J, and Eric F Wieschaus. “Localized requirements for gene activity in segmentation of Drosophila embryos: analysis of armadillo, fused, giant and unpaired mutations in mosaic embryos.”. Roux Arch Dev Biol 195.1 (1986): , 195, 1, 49-62. Web.Abstract
In order to investigate the localized requirements for the activity of genes that are required in a Drosophila embryo for segmentation, we have analyzed the patterns in genetic mosaics. In this paper, we describe our results with four different X-chromosome linked segmentation loci: armadillo, fused, giant and unpaired. For each locus, we first describe in detail the cuticle phenotype of mutant embryos. We then describe the segmentation patterns in embryos mosaic for these mutations, in each case utilizing the shavenbaby (svb) larval cuticle marker mutation to identify the regions of pattern made by genetically mutant cells. For all four loci, we can identify embryos containing large regions of both mutant and wildtype pattern. In these mosaics the regions of mutant pattern are marked with svb and the genetically wildtype (svb (+)) cells make wildtype pattern. The interpretations of the patterns in embryos mosaic for fused and unpaired are complicated by the variability of the phenotypes. However, after taking these complications into account, our principal conclusion is that the requirement for embryonic gene activity seems to be primarily cell autonomous. Based on the descriptions of the mutant phenotypes of these four loci and the analysis of the mosaics, we speculate on the possible roles these genes play in the process of segmentation.
Schüpbach, Trudi, and Eric Wieschaus. “Maternal-effect mutations altering the anterior-posterior pattern of the Drosophila embryo.”. Roux Arch Dev Biol 195.5 (1986): , 195, 5, 302-317. Web.Abstract
Mutations in seven different maternal-effect loci on the second chromosome of Drosophila melanogaster all cause alterations in the anterior-posterior pattern of the embryo. Mutations in torso (tor) and trunk (trk) delete the anterior- and posterior-most structures of the embryo. At the same time they shift cellular fates which are normally found in the subterminal regions of the embryo towards the poles. Mutations in vasa (vas), valois (vls), staufen (stau) and tudor (tud) cause two embryonic defects. For one they result in absence of polar plasm, polar granules and pole cells in all eggs produced by mutant females. Secondly, embryos developing inside such eggs show deletions of abdominal segments. In addition, embryos derived from staufen mothers lack anterior head structures, embryos derived from valois mothers frequently fail to cellularize properly. Mutations in exuperantia (exu) cause deletions of anterior head structures, similar to torso, trunk and staufen. However in exu, these head structures are replaced by an inverted posterior end which comprises posterior midgut, proctodeal region, and often malpighian tubules.The effects of all mutations can be traced back to the beginning stages of gastrulation, indicating that the alterations in cellular fates have probably taken place by that time. Analysis of embryos derived from double mutant mothers suggests that these three phenotypic groups of mutants interfere with three different, independent pathways. All three pathways seem to act additively on the system which specifies anterior-posterior cellular fates within the egg.
Wieschaus, Eric, and Elizabeth Noell. “Specificity of embryonic lethal mutations in Drosophila analyzed in germ line clones.”. Roux Arch Dev Biol 195.1 (1986): , 195, 1, 63-73. Web.Abstract
Only a small fraction of the known mutations causing death to homozygous Drosophila produce gross morphological defects during embryogenesis. We have examined fourteen such loci on the X-chromosome to determine: 1) whether the requirement for their respective activities is restricted to embryogenesis; and 2) whether the embryonic phenotype in mutant embryos is affected by the dosage of wild-type alleles in the mother. For two alleles per locus germ line clones were produced during larval development by irradiating females heterozygous for the lethal mutation and a dominant female sterile (ovo(D)). Only one of the 14 loci (armadillo) is required during development of the germ cell to make morphologically normal eggs. Mutations at two other loci, (bazooka and Notch), allow normal oogenesis but cause major reductions in the viability of genetically normal (i.e., heterozygous) progeny. The majority of the loci (11/14) are not required in the germ line for either oogenesis or embryogenesis. However, in three cases (extradenticle, faintoid and lethal myospheroid), germ line homozygosity results in a readily detectible enhancement of embryonic phenotype over that observed in embryos derived from heterozygous mothers still possessing one wild type allele. The same six loci which show the most substantial effects on germ line homozygosity (arm, baz, N, exd, ftd and mys) also show an amelioration of the mutant phenotypes when maternal dosage is increased to wild type levels by using attached-X females. Four of these same loci (arm, baz, N and exd were cell lethal in imaginal discs.