Early Drosophila Embryo Research Articles

Global shape of Toll activation is determined by wntD enhancer properties

Buffering variability in morphogen distribution is essential for reproducible patterning. A theoretically proposed class of mechanisms, termed "distal pinning," achieves robustness by combining local sensing of morphogen levels with global modulation of gradient spread. Here, we demonstrate a critical role for morphogen sensing by a gene enhancer, which ultimately determines the final global distribution of the morphogen and enables reproducible patterning. Specifically, we show that, while the pattern of Toll activation in the early Drosophila embryo is robust to gene dosage of its locally produced regulator, WntD, it is sensitive to a single-nucleotide change in the wntD enhancer. Thus, enhancer properties of locally produced WntD directly impinge on the global morphogen profile.

The design and logic of terminal patterning in Drosophila.

Terminal regions of the early Drosophila embryo are patterned by the highly conserved ERK cascade, giving rise to the nonsegmented terminal structures of the future larva. In less than an hour, this signaling event establishes several gene expression boundaries and sets in motion a sequence of elaborate morphogenetic events. Genetic studies of terminal patterning discovered signaling components and transcription factors that are involved in numerous developmental contexts and deregulated in human diseases. This review summarizes current understanding of signaling and morphogenesis during terminal patterning and discusses several open questions that can now be rigorously investigated using live imaging, omics, and optogenetic approaches. The anatomical simplicity of the terminal patterning system and its amenability to a broad range of increasingly sophisticated genetic perturbations will continue to make it a premier quantitative model for studying multiple aspects of tissue patterning by dynamically controlled cell signaling pathways.

A matter of time: Formation and interpretation of the Bicoid morphogen gradient.

Spatially distributed signaling molecules, known as morphogens, provide spatial information during development. A host of different morphogens have now been identified, from subcellular gradients through to morphogens that act across a whole embryo. These gradients form over a wide-range of timescales, from seconds to hours, and their time windows for interpretation are also highly variable; the processes of morphogen gradient formation and interpretation are highly dynamic. The morphogen Bicoid (Bcd), present in the early Drosophila embryo, is essential for setting up the future Drosophila body segments. Due to its accessibility for both genetic perturbations and imaging, this system has provided key insights into how precise patterning can occur within a highly dynamic system. Here, we review the temporal scales of Bcd gradient formation and interpretation. In particular, we discuss the quantitative evidence for different models of Bcd gradient formation, outline the time windows for Bcd interpretation, and describe how Bcd temporally adapts its own ability to be interpreted. The utilization of temporal information in morphogen readout may provide crucial inputs to ensure precise spatial patterning, particularly in rapidly developing systems.

Formation, interpretation, and regulation of the Drosophila Dorsal/NF-κB gradient.

The morphogen gradient of the transcription factor Dorsal in the early Drosophila embryo has become one of the most widely studied tissue patterning systems. Dorsal is a Drosophila homolog of mammalian NF-κB and patterns the dorsal-ventral axis of the blastoderm embryo into several tissue types by spatially regulating upwards of 100 zygotic genes. Recent studies using fluorescence microscopy and live imaging have quantified the Dorsal gradient and its target genes, which has paved the way for mechanistic modeling of the gradient. In this review, we describe the mechanisms behind the initiation of the Dorsal gradient and its regulation of target genes. The main focus of the review is a discussion of quantitative and computational studies of the Dl gradient system, including regulation of the Dl gradient. We conclude with a discussion of potential future directions.

Microtubules are necessary for proper Reticulon localization during mitosis.

During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. Here, we examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, we show that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, we show that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle.

Coacting enhancers can have complementary functions within gene regulatory networks and promote canalization.

Developmental genes are often regulated by multiple enhancers exhibiting similar spatiotemporal outputs, which are generally considered redundantly acting though few have been studied functionally. Using CRISPR-Cas9, we created deletions of two enhancers, brk5’ and brk3’, that drive similar but not identical expression of the gene brinker (brk) in early Drosophila embryos. Utilizing both in situ hybridization and quantitative mRNA analysis, we investigated the changes in the gene network state caused by the removal of one or both of the early acting enhancers. brk5’ deletion generally phenocopied the gene mutant, including expansion of the BMP ligand decapentaplegic (dpp) as well as inducing variability in amnioserosa tissue cell number suggesting a loss of canalization. In contrast, brk3’ deletion presented unique phenotypes including dorsal expansion of several ventrally expressed genes and a decrease in amnioserosa cell number. Similarly, deletions were made for two enhancers associated with the gene short-gastrulation (sog), sog.int and sog.dist, demonstrating that they also exhibit distinct patterning phenotypes and affect canalization. In summary, this study shows that similar gene expression driven by coacting enhancers can support distinct, and sometimes complementary, functions within gene regulatory networks and, moreover, that phenotypes associated with individual enhancer deletion mutants can provide insight into new gene functions.

Protein components of ribonucleoprotein granules from Drosophila germ cells oligomerize and show distinct spatial organization during germline development

The assembly of large RNA-protein granules occurs in germ cells of many animals and these germ granules have provided a paradigm to study structure-functional aspects of similar structures in different cells. Germ granules in Drosophila oocyte’s posterior pole (polar granules) are composed of RNA, in the form of homotypic clusters, and proteins required for germline development. In the granules, Piwi protein Aubergine binds to a scaffold protein Tudor, which contains 11 Tudor domains. Using a super-resolution microscopy, we show that surprisingly, Aubergine and Tudor form distinct clusters within the same polar granules in early Drosophila embryos. These clusters partially overlap and, after germ cells form, they transition into spherical granules with the structural organization unexpected from these interacting proteins: Aubergine shell around the Tudor core. Consistent with the formation of distinct clusters, we show that Aubergine forms homo-oligomers and using all purified Tudor domains, we demonstrate that multiple domains, distributed along the entire Tudor structure, interact with Aubergine. Our data suggest that in polar granules, Aubergine and Tudor are assembled into distinct phases, partially mixed at their “interaction hubs”, and that association of distinct protein clusters may be an evolutionarily conserved mechanism for the assembly of germ granules.

Functional analysis of engrailed in Tribolium segmentation

The segment-polarity gene engrailed is required for segmentation in the early Drosophila embryo. Loss of Engrailed function results in segmentation defects that vary in severity from pair-rule phenotypes to a lawn phenotype lacking in obvious of segmentation. During segmentation, Engrailed is expressed in stripes with a single segmental periodicity in Drosophila, which is conserved in all arthropods examined so far. To define segments, the segmental stripes of Engrailed induce the segmental stripes of wingless at each parasegmental boundary. However, segmentation functions of orthologs of engrailed in non-Drosophila arthropods have yet to be reported. Here, we analyzed functions of the Tribolium ortholog of engrailed (Tc-engrailed) during embryonic segmentation. Larval cuticles with Tc-engrailed being knocked down had segmentation phenotypes including incomplete segment formation and loss of a group of segments. In agreement with the cuticle segmentation defects, segments developed incompletely and irregularly or did not form in Tribolium germbands where Tc-engrailed was knocked down. Furthermore, knock-down of Tc-engrailed did not properly express the segmental stripes of wingless in Tribolium germbands. Taken together with the conserved expression patterns of Engrailed in arthropod segmentation, our data suggest that Tc-engrailed is required for embryonic segmentation in Tribolium, and the genetic mechanism of Engrailed inducing wingless expression is conserved at least between Drosophila and Tribolium.

Dynamics of Spaetzle morphogen shuttling in the Drosophila embryo shapes gastrulation patterning.

Establishment of morphogen gradients in the early Drosophila embryo is challenged by a diffusible sextracellular milieu, and by rapid nuclear divisions that occur at the same time. To understand how a sharp gradient is formed within this dynamic environment, we followed the generation of graded nuclear Dorsal protein, the hallmark of pattern formation along the dorso-ventral axis, in live embryos. The dynamics indicate that a sharp extracellular gradient is formed through diffusion-based shuttling of the Spaetzle (Spz) morphogen that progresses through several nuclear divisions. Perturbed shuttling in wntD mutant embryos results in a flat activation peak and aberrant gastrulation. Re-entry of Dorsal into the nuclei at the final division cycle plays an instructive role, as the residence time of Dorsal in each nucleus is translated to the amount of zygotic transcript that will be produced, thereby guiding graded accumulation of specific zygotic transcripts that drive patterned gastrulation. We conclude that diffusion-based ligand shuttling, coupled with dynamic readout, establishes a refined pattern within the diffusible environment of early embryos.

The genome-wide multi-layered architecture of chromosome pairing in early Drosophila embryos

Genome organization involves cis and trans chromosomal interactions, both implicated in gene regulation, development, and disease. Here, we focus on trans interactions in Drosophila, where homologous chromosomes are paired in somatic cells from embryogenesis through adulthood. We first address long-standing questions regarding the structure of embryonic homolog pairing and, to this end, develop a haplotype-resolved Hi-C approach to minimize homolog misassignment and thus robustly distinguish trans-homolog from cis contacts. This computational approach, which we call Ohm, reveals pairing to be surprisingly structured genome-wide, with trans-homolog domains, compartments, and interaction peaks, many coinciding with analogous cis features. We also find a significant genome-wide correlation between pairing, transcription during zygotic genome activation, and binding of the pioneer factor Zelda. Our findings reveal a complex, highly structured organization underlying homolog pairing, first discovered a century ago in Drosophila. Finally, we demonstrate the versatility of our haplotype-resolved approach by applying it to mammalian embryos.

The cellular and molecular mechanisms that establish the mechanics of Drosophila gastrulation.

In this review, we cover advances in the field that have contributed to our mechanistic understanding of how tissues internalize during Drosophila melanogaster gastrulation. The changes in tissue shape and architecture that are associated with mesoderm and endoderm invagination in the early Drosophila embryo are accompanied by cell shape changes which are driven by actomyosin contractility. The activation of signal transduction pathways is patterned by embryonic transcription factors, which define distinct geometries of gene expression and the tissue contractile domains. At the subcellular level, outputs from signaling pathways that activate actomyosin contractility are highly polarized and their behavior is fine-tuned by a balance of both positive and negative regulation. Cells are mechanically linked through adherens junctions, allowing forces that are generated by cells to be integrated across the tissue, ensuring coordinated cell behavior during tissue invagination. The transmission of force between cells also enables mechanical feedback whereby force generation influences both cell and cytoskeletal behavior. Finally, after tissue invagination, mesoderm cells undergo an epithelial-to-mesenchymal transition and cell spreading. We highlight studies that have utilized this model system to uncover fundamental principles at molecular-, cell-, and tissue-levels, which have contributed to our understanding of similar tissue morphogenetic processes across different organisms.

Self-Similar Dynamics of Nuclear Packing in the Early Drosophila Embryo

Embryonic development starts with cleavages, a rapid sequence of reductive divisions that result in an exponential increase of cell number without changing the overall size of the embryo. In Drosophila, the final four rounds of cleavages occur at the surface of the embryo and give rise to ∼6000 nuclei under a common plasma membrane. We use live imaging to study the dynamics of this process and to characterize the emergent nuclear packing in this system. We show that the characteristic length scale of the internuclear interaction scales with the density, which allows the densifying embryo to sustain the level of structural order at progressively smaller length scales. This is different from nonliving materials, which typically undergo disorder-order transition upon compression. To explain this dynamics, we use a particle-based model that accounts for density-dependent nuclear interactions and synchronous divisions. We reproduce the pair statistics of the disordered packings observed in embryos and recover the scaling relation between the characteristic length scale and the density both in real and reciprocal space. This result reveals how the embryo can robustly preserve the nuclear-packing structure while being densified. In addition to providing quantitative description of self-similar dynamics of nuclear packings, this model generates dynamic meshes for the computational analysis of pattern formation and tissue morphogenesis.

Distinct Roles of Broadly Expressed Repressors Support Dynamic Enhancer Action and Change in Time

SUMMARYHow broadly expressed repressors regulate gene expression is incompletely understood. To gain insight, we investigated how Suppressor of Hairless—Su(H)—and Runt regulate expression of bone morphogenetic protein (BMP) antagonist short-gastrulation via the sog_Distal enhancer. A live imaging protocol was optimized to capture this enhancer’s spatiotemporal output throughout the early Drosophila embryo, finding in this context that Runt regulates transcription initiation, Su(H) regulates transcription rate, and both factors control spatial expression. Furthermore, whereas Su(H) functions as a dedicated repressor, Runt temporally switches from repressor to activator. Our results demonstrate that broad repressors play temporally distinct roles and contribute to dynamic gene expression. Both Run and Su(H)’s ability to influence the spatiotemporal domains of gene expression may serve to counterbalance activators and function in this manner as important regulators of the maternal-to-zygotic transition in early embryos.

6mA-DNA-binding factor Jumu controls maternal-to-zygotic transition upstream of Zelda

A long-standing question in the field of embryogenesis is how the zygotic genome is precisely activated by maternal factors, allowing normal early embryonic development. We have previously shown that N6-methyladenine (6mA) DNA modification is highly dynamic in early Drosophila embryos and forms an epigenetic mark. However, little is known about how 6mA-formed epigenetic information is decoded. Here we report that the Fox-family protein Jumu binds 6mA-marked DNA and acts as a maternal factor to regulate the maternal-to-zygotic transition. We find that zelda encoding the pioneer factor Zelda is marked by 6mA. Our genetic assays suggest that Jumu controls the proper zygotic genome activation (ZGA) in early embryos, at least in part, by regulating zelda expression. Thus, our findings not only support that the 6mA-formed epigenetic marks can be read by specific transcription factors, but also uncover a mechanism by which the Jumu regulates ZGA partially through Zelda in early embryos.

Nuclear (Bio)physics in the Embryo

Deneke etal. (2019) discover that dynamic interactions of cell cycle and actomyosin contractility systems synchronize nuclear cleavages, generating a cytoplasmic flow that results in a spatially uniform distribution of zygotic nuclei in the early Drosophila embryo. This work underscores the importance of self-organizing mechanisms before the onset of zygotic transcription.

The Drosophila Pioneer Factor Zelda Modulates the Nuclear Microenvironment of a Dorsal Target Enhancer to Potentiate Transcriptional Output

Connecting the developmental patterning of tissues to the mechanistic control of RNA polymerase II remains a long-term goal of developmental biology. Many key elements have been identified in the establishment of spatial-temporal control of transcription in the early Drosophila embryo, a model system for transcriptional regulation. The dorsal-ventral axis of the Drosophila embryo is determined by the graded distribution of Dorsal (Dl), a homolog of the nuclear factor κB (NF-κB) family of transcriptional activators found in humans [1, 2]. A second maternally deposited factor, Zelda (Zld), is uniformly distributed in the embryo and is thought to act as a pioneer factor, increasing enhancer accessibility for transcription factors, such as Dl [3-9]. Here, we utilized the MS2 live imaging system to evaluate the expression of the Dl target gene short gastrulation (sog) to better understand how a pioneer factor affects the kinetic parameters of transcription. Our experiments indicate that Zld modifies probability of activation, the timing of this activation, and the rate at which transcription occurs. Our results further show that this effective rate increase is due to an increased accumulation of Dl at the site of transcription, suggesting that transcription factor "hubs" induced by Zld [10] functionally regulate transcription.

A Link between Deoxyribonucleotide Metabolites and Embryonic Cell-Cycle Control

The egg contains maternal RNAs and proteins, whichhave instrumental functions in patterning and morphogenesis. Besides these, the egg also contains metabolites, whose developmental functions have been little investigated. For example, the rapid increase of DNA content during the fast embryonic cell cycles poses high demands on the supply of deoxyribonucleotides (dNTPs), which may be synthesized in the embryo or maternally provided [1, 2]. Here, we analyze the role of dNTP in early Drosophila embryos. We found that dNTP levels initially decreased about 2-fold before reaching stable levels at the transition from syncytial to cellular blastoderm. Employing a mutant of the metabolic enzyme serinehydroxymethyl transferase (SHMT), which isimpaired in the embryonic synthesis of deoxythymidine triphosphate (dTTP), we found that the maternal supply of dTTP was specifically depleted by interphase 13. SHMT mutants showed persistent Sphase, replication stress, and a checkpoint-dependentcell-cycle arrest in NC13, depending on the loss of dTTP. The cell-cycle arrest in SHMT mutants was suppressed by reduced zygotic transcription. Consistent with the requirement of dTTP for cell-cycle progression, increased dNTP levels accelerated the cell cycle in embryos lacking zygotic transcription. We propose a model that both a limiting dNTP supply and interference of zygotic transcription with DNA replication [3] elicit DNA replication stress and checkpoint activation. Our study reveals a specific mechanism of how dNTP metabolites contribute to the embryonic cell-cycle control.

A Role for buttonhead in the Early Head and Trunk Development in the Beetle Tribolium castaneum.

Thehead gap gene buttonhead (btd) is required for the patterning of head segments in the early Drosophila embryo. Mutant phenotypes of btd display a gap-like phenotype in which antennal, intercalary, mandibular and the anterior portion of the maxillary segmentsare eliminated. In agreement with the phenotypes, btd is expressed in a stripe covering the head segments at the blastoderm stage. During the early phase of the germband extension, btd is expressed in stripes with single segmental periodicity, which is required for the formation of the peripheral nervous system. In contrast to the key role of btd in Drosophila embryonic development, it has been suggested that Tribolium ortholog of btd (Tc-btd) is dispensable for embryonic head development. In order for better understanding of the requirement of Tc-btd in the early Tribolium embryo, we re-analyzed the expression patterns and functions of Tc-btd during embryonic segmentation. Tc-btd is expressed in segmental stripes at the stages of blastoderm and germband elongation. Up to 28.3% of embryos in which Tc-btd is knocked down displays the loss of antennal, mandibular and the pregnathal regions in the head, with abdominal segments being disrupted in the trunk. Our findings suggest that Tc-btd is required for the head and trunk development in the early Tribolium embryo.

Rapid embryonic cell cycles defer the establishment of heterochromatin by Eggless/SetDB1 in Drosophila.

Acquisition of chromatin modifications during embryogenesis distinguishes different regions of an initially naïve genome. In many organisms, repetitive DNA is packaged into constitutive heterochromatin that is marked by di/trimethylation of histone H3K9 and the associated protein HP1a. These modifications enforce the unique epigenetic properties of heterochromatin. However, in the early Drosophila melanogaster embryo, the heterochromatin lacks these modifications, which appear only later, when rapid embryonic cell cycles slow down at the midblastula transition (MBT). Here we focus on the initial steps restoring heterochromatic modifications in the embryo. We describe the JabbaTrap, a technique for inactivating maternally provided proteins in embryos. Using the JabbaTrap, we reveal a major requirement for the methyltransferase Eggless/SetDB1 in the establishment of heterochromatin. In contrast, other methyltransferases contribute minimally. Live imaging reveals that endogenous Eggless gradually accumulates on chromatin in interphase but then dissociates in mitosis, and its accumulation must restart in the next cell cycle. Cell cycle slowing as the embryo approaches the MBT permits increasing accumulation and action of Eggless at its targets. Experimental manipulation of interphase duration shows that cell cycle speed regulates Eggless. We propose that developmental slowing of the cell cycle times embryonic heterochromatin formation.

Signaling Dynamics Control Cell Fate in the Early Drosophila Embryo

The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? We combine optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. We find that Erk activity is sufficient to "posteriorize" 88% of the embryo, inducing gut endoderm-like gene expression and morphogenetic movements in all cells within this region. Gut endoderm fate adoption requires at least 1h of signaling, whereas a 30-min Erk pulse specifies a distinct ectodermal cell type, intermediate neuroblasts. We find that the endoderm-ectoderm cell fate switch is controlled by the cumulative load of Erk activity, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profileof Erksignaling selects between distinct physiological outcomes.