Setting the stage for development: the maternal-to-zygotic transition in Drosophila.

The zygote has a daunting task ahead of itself; it must develop from a single cell (fertilized egg) into a fully functioning adult with a multitude of different cell types. In the beginning, the zygote has help from its mother, in the form of gene products deposited into the egg, but eventually, it must rely on its own resources to proceed through development. The transfer of developmental control from the mother to the embryo is called the maternal-to-zygotic transition (MZT). All animals undergo this transition, which is defined by two main processes-the degradation of maternal RNAs and the synthesis of new RNAs from the zygote's own genome. Here, we review the regulation of the MZT in Drosophila, but given the broad conservation of this essential process, much of the regulation is shared among metazoans.

Chromatin accessibility in the Drosophila embryo is determined by transcription factor pioneering and enhancer activation.

Chromatin accessibility is integral to the process by which transcription factors (TFs) read out cis-regulatory DNA sequences, but it is difficult to differentiate between TFs that drive accessibility and those that do not. Deep learning models that learn complex sequence rules provide an unprecedented opportunity to dissect this problem. Using zygotic genome activation in Drosophila as a model, we analyzed high-resolution TF binding and chromatin accessibility data with interpretable deep learning and performed genetic validation experiments. We identify a hierarchical relationship between the pioneer TF Zelda and the TFs involved in axis patterning. Zelda consistently pioneers chromatin accessibility proportional to motif affinity, whereas patterning TFs augment chromatin accessibility in sequence contexts where they mediate enhancer activation. We conclude that chromatin accessibility occurs in two tiers: one through pioneering, which makes enhancers accessible but not necessarily active, and the second when the correct combination of TFs leads to enhancer activation.

Enhancer architecture and chromatin accessibility constrain phenotypic space during Drosophila development.

Developmental enhancers bind transcription factors and dictate patterns of gene expression during development. Their molecular evolution can underlie phenotypical evolution, but the contributions of the evolutionary pathways involved remain little understood. Here, using mutation libraries in Drosophila melanogaster embryos, we observed that most point mutations in developmental enhancers led to changes in gene expression levels but rarely resulted in novel expression outside of the native pattern. In contrast, random sequences, often acting as developmental enhancers, drove expression across a range of cell types; random sequences including motifs for transcription factors with pioneer activity acted as enhancers even more frequently. Our findings suggest that the phenotypic landscapes of developmental enhancers are constrained by enhancer architecture and chromatin accessibility. We propose that the evolution of existing enhancers is limited in its capacity to generate novel phenotypes, whereas the activity of de novo elements is a primary source of phenotypic novelty.

Shadow enhancers modulate distinct transcriptional parameters that differentially effect downstream patterning events.

Transcription in the early Drosophila blastoderm is coordinated by the collective action of hundreds of enhancers. Many genes are controlled by so-called 'shadow enhancers', which provide resilience to environment or genetic insult, allowing the embryo to robustly generate a precise transcriptional pattern. Emerging evidence suggests that many shadow enhancer pairs do not drive identical expression patterns, but the biological significance of this remains unclear. In this study, we characterize the shadow enhancer pair controlling the gene short gastrulation (sog). We removed either the intronic proximal enhancer or the upstream distal enhancer and monitored sog transcriptional kinetics. Notably, each enhancer differs in sog spatial expression, timing of activation and RNA Polymerase II loading rates. In addition, modeling of individual enhancer activities demonstrates that these enhancers integrate activation and repression signals differently. Whereas activation is due to the sum of the two enhancer activities, repression appears to depend on synergistic effects between enhancers. Finally, we examined the downstream signaling consequences resulting from the loss of either enhancer, and found changes in tissue patterning that can be explained by the differences in transcriptional kinetics measured.

Spatial organization of transcribing loci during early genome activation in Drosophila.

The early Drosophila embryo provides unique experimental advantages for addressing fundamental questions of gene regulation at multiple levels of organization, from individual gene loci to the entire genome. Using 1.5-h-old Drosophila embryos undergoing the first wave of genome activation,1 we detected ∼110 discrete "speckles" of RNA polymerase II (RNA Pol II) per nucleus, two of which were larger and localized to the histone locus bodies (HLBs).2,3 In the absence of the primary driver of Drosophila genome activation, the pioneer factor Zelda (Zld),1,4,5 70% fewer speckles were present; however, the HLBs tended to be larger than wild-type (WT) HLBs, indicating that RNA Pol II accumulates at the HLBs in the absence of robust early-gene transcription. We observed a uniform distribution of distances between active genes in the nuclei of both WT and zld mutant embryos, indicating that early co-regulated genes do not cluster into nuclear sub-domains. However, in instances whereby transcribing genes did come into close 3D proximity (within 400 nm), they were found to have distinct RNA Pol II speckles. In contrast to the emerging model whereby active genes are clustered to facilitate co-regulation and sharing of transcriptional resources, our data support an "individualist" model of gene control at early genome activation in Drosophila. This model is in contrast to a "collectivist" model, where active genes are spatially clustered and share transcriptional resources, motivating rigorous tests of both models in other experimental systems.

Capicua is a fast-acting transcriptional brake.

Even though transcriptional repressors are studied with ever-increasing molecular resolution, the temporal aspects of gene repression remain poorly understood. Here, we address the dynamics of transcriptional repression by Capicua (Cic), which is essential for normal development and is commonly mutated in human cancers and neurodegenerative diseases.1,2 We report the speed limit for Cic-dependent gene repression based on live imaging and optogenetic perturbations in the early Drosophila embryo, where Cic was originally discovered.3 Our measurements of Cic concentration and intranuclear mobility, along with real-time monitoring of the activity of Cic target genes, reveal remarkably fast transcriptional repression within minutes of removing an optogenetic de-repressive signal. In parallel, quantitative analyses of transcriptional bursting of Cic target genes support a repression mechanism providing a fast-acting brake on burst generation. This work sets quantitative constraints on potential mechanisms for gene regulation by Cic.

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.

Metabolic Regulation of Developmental Cell Cycles and Zygotic Transcription.

The thirteen nuclear cleavages that give rise to the Drosophila blastoderm are some of the fastest known cell cycles [1]. Surprisingly, the fertilized egg is provided with at most one-third of the dNTPs needed to complete the thirteen rounds of DNA replication [2]. The rest must be synthesized by the embryo, concurrent with cleavage divisions. What is the reason for the limited supply of DNA building blocks? We propose that frugal control of dNTP synthesis contributes to the well-characterized deceleration of the cleavage cycles and is needed for robust accumulation of zygotic gene products. In support of this model, we demonstrate that when the levels of dNTPs are abnormally high, nuclear cleavages fail to sufficiently decelerate, the levels of zygotic transcription are dramatically reduced, and the embryo catastrophically fails early in gastrulation. Our work reveals a direct connection between metabolism, the cell cycle, and zygotic transcription.

BMP Signaling Determines Body Size via Transcriptional Regulation of Collagen Genes in Caenorhabditis elegans.

Body size is a tightly regulated phenotype in metazoans that depends on both intrinsic and extrinsic factors. While signaling pathways are known to control organ and body size, the downstream effectors that mediate their effects remain poorly understood. In the nematode Caenorhabditis elegans, a Bone Morphogenetic Protein (BMP)-related signaling pathway is the major regulator of growth and body size. We investigated the transcriptional network through which the BMP pathway regulates body size and identified cuticle collagen genes as major effectors of growth control. We demonstrate that cuticle collagens can act as positive regulators (col-41), negative regulators (col-141), or dose-sensitive regulators (rol-6) of body size. Moreover, we find a requirement of BMP signaling for stage-specific expression of cuticle collagen genes. We show that the Smad signal transducers directly bind conserved Smad-binding elements in regulatory regions of col-141 and col-142, but not of col-41 Hence, cuticle collagen genes may be directly and indirectly regulated via the BMP pathway. Our work thus connects a conserved signaling pathway with its critical downstream effectors, advancing insight into how body size is specified. Since collagen mutations and misregulation are implicated in numerous human genetic disorders and injury sequelae, understanding how collagen gene expression is regulated has broad implications.

A Systematic Ensemble Approach to Thermodynamic Modeling of Gene Expression from Sequence Data.

To understand the relationship between an enhancer DNA sequence and quantitative gene expression, thermodynamics-driven mathematical models of transcription are often employed. These "sequence-to-expression" models can describe an incomplete or even incorrect set of regulatory relationships if the parameter space is not searched systematically. Here, we focus on an enhancer of the Drosophila gene ind and demonstrate how a systematic search of parameter space can reveal a more comprehensive picture of a gene's regulatory mechanisms, resolve outstanding ambiguities, and suggest testable hypotheses. We describe an approach that generates an ensemble of ind models; all of these models are technically acceptable solutions to the sequence-to-expression problem in light of wild-type data, and some represent mechanistically distinct hypotheses about the regulation of ind. This ensemble can be restricted to biologically plausible models using requirements gleaned from in vivo perturbation experiments. Biologically plausible models make unique predictions about how specific ind enhancer sequences affect ind expression; we validate these predictions in vivo through site mutagenesis in transgenic Drosophila embryos.

Zelda potentiates morphogen activity by increasing chromatin accessibility.

Zygotic genome activation (ZGA) is a major genome programming event whereby the cells of the embryo begin to adopt specified fates. Experiments in Drosophila and zebrafish have revealed that ZGA depends on transcription factors that provide large-scale control of gene expression by direct and specific binding to gene regulatory sequences. Zelda (Zld) plays such a role in the Drosophila embryo, where it has been shown to control the action of patterning signals; however, the mechanisms underlying this effect remain largely unclear. A recent model proposed that Zld binding sites act as quantitative regulators of the spatiotemporal expression of genes activated by Dorsal (Dl), the morphogen that patterns the dorsoventral axis. Here we tested this model experimentally, using enhancers of brinker (brk) and short gastrulation (sog), both of which are directly activated by Dl, but at different concentration thresholds. In agreement with the model, we show that there is a clear positive correlation between the number of Zld binding sites and the spatial domain of enhancer activity. Likewise, the timing of expression could be advanced or delayed. We present evidence that Zld facilitates binding of Dl to regulatory DNA, and that this is associated with increased chromatin accessibility. Importantly, the change in chromatin accessibility is strongly correlated with the change in Zld binding, but not Dl. We propose that the ability of genome activators to facilitate readout of transcriptional input is key to widespread transcriptional induction during ZGA.

Temporal dynamics, spatial range, and transcriptional interpretation of the Dorsal morphogen gradient.

Dorsoventral pattern of Drosophila embryo is specified by the nuclear localization gradient of the transcription factor Dorsal. Genetic and genomic studies of this morphogen gradient provided important insights into spatial control of gene expression in development. Recent live imaging experiments revealed hitherto unappreciated dynamics of the Dorsal gradient and posed new questions about the mechanisms of its transcriptional interpretation. Some of these questions can be answered by models in which the morphogenetic capacity of the Dorsal gradient is potentiated by spatially uniform factors, such as Zelda, a transcription factor that plays a key role in the activation of zygotic transcription. Combinatorial effects of uniform and graded factors play an important role in the transcriptional and signaling cascades initiated by Dorsal and may explain differential positioning of gene expression borders by other morphogen gradients.

Pattern formation by graded and uniform signals in the early Drosophila embryo.

The early Drosophila embryo is patterned by graded distributions of maternal transcription factors. Recent studies revealed that pattern formation by these graded signals depends on uniformly expressed transcriptional activators, such as Zelda. Removal of Zelda influences both the timing and the spatial expression domains for most of the genes controlled by maternal gradients. We demonstrate that some of these patterning defects, which range from temporal delay to loss of expression, can be rationalized with the use of a mathematical model based on cooperative binding of graded and uniform factors. This model makes a number of predictions, which we confirm experimentally by analyzing the expression of short gastrulation (sog), a gene that is controlled by a combination of the Dorsal morphogen gradient and Zelda. The proposed model suggests a general mechanism for the formation of nested gene expression domains, which is a hallmark of tissue patterning by morphogen gradients. According to this mechanism, the differential effects of a morphogen on its target genes can depend on their differential sensitivity to uniform factors.