Early Drosophila Embryo Research Articles

HIRA and dPCIF1 coordinately establish totipotent chromatin and control orderly ZGA in Drosophila embryos

Early embryos undergo profound changes in their genomic architecture to establish the totipotent state, enabling pioneer factors to access chromatin and drive zygotic genome activation (ZGA). However, the mechanisms by which the totipotent state is established and properly interpreted by pioneer factors to allow orderly ZGA remain unknown. Here, we identify the H3.3-specific chaperone HIRA as a factor involving establishing totipotent-state chromatin in Drosophila early embryos. Through cophase separation with HIRA, the pioneer factor GAGA factor (GAF) efficiently binds to H3.3-marked nucleosomes to activate major-wave zygotic genes. Importantly, dPCIF1, a chromatin-associated protein, antagonized the GAF-HIRA interaction by competitively binding to HIRA, thereby restricting GAF on earlier chromatin and avoiding premature ZGA. Hence, the coordinated action of HIRA and dPCIF1 ensures sequential ZGA from the minor to major wave in early embryos. This study provides insights into understanding how a totipotent state is established and properly controlled during ZGA.

Su(H) Modulates Enhancer Transcriptional Bursting in Prelude to Gastrulation.

Transcriptional regulation, orchestrated by the interplay between transcription factors (TFs) and enhancers, governs gene expression dynamics crucial for cellular processes. While gross qualitative fluctuations in transcription factor-dependent gene expression patterning have a long history of characterization, the roles of these factors in the nuclei retaining expression in the presence or absence of these factors are now observable using modern techniques. Our study investigates the impact of Suppressor of Hairless (Su(H)), a broadly expressed transcription factor, on enhancer-driven transcriptional modulation using Drosophila early embryos as a model system. Building upon previous findings, we employ super-resolution microscopy to dissect Su(H)'s influence on sog-Distal (sogD) enhancer activity specifically in nuclei with preserved sogD-driven expression in the absence of Su(H) binding. We demonstrate that Su(H) occupancy perturbations alter expression levels and bursting dynamics. Notably, Su(H) absence during embryonic development exhibits region-specific effects, inhibiting expression dorsally and stabilizing expression ventrally, implying a nuanced role in enhancer regulation. Our findings shed light on the intricate mechanisms that govern transcriptional dynamics and suggest a critical patterning role for Notch/Hairless signaling in sog expression as embryos transition to gastrulation.

Widespread regulation of the maternal transcriptome by Nanos in Drosophila.

The translational repressor Nanos (Nos) regulates a single target, maternal hunchback (hb) mRNA, to govern abdominal segmentation in the early Drosophila embryo. Nos is recruited to sites in the 3' UTR of hb mRNA in collaboration with the sequence-specific RNA-binding protein Pumilio (Pum); on its own, Nos has no binding specificity. Nos is expressed at other stages of development, but very few mRNA targets that might mediate its action at these stages have been described. Nor has it been clear whether Nos is targeted to other mRNAs in concert with Pum or via other mechanisms. In this report, we identify mRNAs targeted by Nos via 2 approaches. First, we identify mRNAs depleted upon expression of a chimera bearing Nos fused to the nonsense mediated decay (NMD) factor Upf1. We find that, in addition to hb, Upf1-Nos depletes approximately 2,600 mRNAs from the maternal transcriptome in early embryos. Virtually all of these appear to be targeted in a canonical, hb-like manner in concert with Pum. In a second, more conventional approach, we identify mRNAs that are stabilized during the maternal zygotic transition (MZT) in embryos from nos- females. Most (86%) of the 1,185 mRNAs regulated by Nos are also targeted by Upf1-Nos, validating use of the chimera. Previous work has shown that 60% of the maternal transcriptome is degraded in early embryos. We find that maternal mRNAs targeted by Upf1-Nos are hypoadenylated and inefficiently translated at the ovary-embryo transition; they are subsequently degraded in the early embryo, accounting for 59% of all destabilized maternal mRNAs. We suggest that the late ovarian burst of Nos represses a large fraction of the maternal transcriptome, priming it for later degradation by other factors in the embryo.

Differential regulation of the proteome and phosphoproteome along the dorso-ventral axis of the early Drosophila embryo.

The initially homogeneous epithelium of the early Drosophila embryo differentiates into regional subpopulations with different behaviours and physical properties that are needed for morphogenesis. The factors at top of the genetic hierarchy that control these behaviours are known, but many of their targets are not. To understand how proteins work together to mediate differential cellular activities, we studied in an unbiased manner the proteomes and phosphoproteomes of the three main cell populations along the dorso-ventral axis during gastrulation using mutant embryos that represent the different populations. We detected 6111 protein groups and 6259 phosphosites of which 3398 and 3433 were differentially regulated, respectively. The changes in phosphosite abundance did not correlate with changes in host protein abundance, showing phosphorylation to be a regulatory step during gastrulation. Hierarchical clustering of protein groups and phosphosites identified clusters that contain known fate determinants such as Doc1, Sog, Snail, and Twist. The recovery of the appropriate known marker proteins in each of the different mutants we used validated the approach, but also revealed that two mutations that both interfere with the dorsal fate pathway, Toll10B and serpin27aex do this in very different manners. Diffused network analyses within each cluster point to microtubule components as one of the main groups of regulated proteins. Functional studies on the role of microtubules provide the proof of principle that microtubules have different functions in different domains along the DV axis of the embryo.

Dynamics of BMP signaling and stable gene expression in the early Drosophila embryo.

In developing tissues, morphogen gradients are thought to initialize gene expression patterns. However, the relationship between the dynamics of morphogen-encoded signals and gene expression decisions is largely unknown. Here we examine the dynamics of the Bone Morphogenetic Protein (BMP) pathway in Drosophila blastoderm-stage embryos. In this tissue, the BMP pathway is highly dynamic: it begins as a broad and weak signal on the dorsal half of the embryo, then 20-30 min later refines into a narrow, intense peak centered on the dorsal midline. This dynamical progression of the BMP signal raises questions of how it stably activates target genes. Therefore, we performed live imaging of the BMP signal and found that dorsal-lateral cells experience only a short transient in BMP signaling, after which the signal is lost completely. Moreover, we measured the transcriptional response of the BMP target gene pannier in live embryos and found it to remain activated in dorsal-lateral cells, even after the BMP signal is lost. Our findings may suggest that the BMP pathway activates a memory, or 'ratchet' mechanism that may sustain gene expression.

H3K14ac facilitates the reinstallation of constitutive heterochromatin in Drosophila early embryos by engaging Eggless/SetDB1

Constitutive heterochromatin, a fundamental feature of eukaryotic nucleus essential for transposon silencing and genome stability, is rebuilt on various types of repetitive DNA in the zygotic genome during early embryogenesis. However, the molecular program underlying this process remains poorly understood. Here, we show that histone H3 lysine 14 acetylation (H3K14ac) is engaged in the reinstallation of constitutive heterochromatin in Drosophila early embryos. H3K14ac partially colocalizes with H3 lysine 9 trimethylation (H3K9me3) and its methyltransferase Eggless/SetDB1 around the mid-blastula transition. Concealing H3K14ac by either antibody injection or maternal knockdown of Gcn5 diminishes Eggless/SetDB1 nuclear foci and reduces the deposition of H3K9me3. Structural analysis reveals that Eggless/SetDB1 recognizes H3K14ac via its tandem Tudor domains, and disrupting the binding interface causes defects in Eggless/SetDB1 distribution and derepression of a subset of transposons. Therefore, H3K14ac, a histone modification normally associated with active transcription, is a crucial component of the early embryonic machinery that introduces constitutive heterochromatic features to the newly formed zygotic genome.

Both the transcriptional activator, Bcd, and repressor, Cic, form small mobile oligomeric clusters

Transcription factors play an essential role in pattern formation during early embryo development, generating a strikingly fast and precise transcriptional response that results in sharp gene expression boundaries. To characterize the steps leading up to transcription, we performed a side-by-side comparison of the nuclear dynamics of two morphogens, a transcriptional activator, Bicoid (Bcd), and a transcriptional repressor, Capicua (Cic), both involved in body patterning along the anterior-posterior axis of the early Drosophila embryo. We used a combination of fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and single-particle tracking to access a wide range of dynamical timescales. Despite their opposite effects on gene transcription, we find that Bcd and Cic have very similar nuclear dynamics, characterized by the coexistence of a freely diffusing monomer population with a number of oligomeric clusters, which range from low stoichiometry and high mobility clusters to larger, DNA-bound hubs. Our observations are consistent with the inclusion of both Bcd and Cic into transcriptional hubs or condensates, while putting constraints on the mechanism by which these form. These results fit in with the recent proposal that many transcription factors might share a common search strategy for target gene regulatory regions that makes use of their large unstructured regions, and may eventually help explain how the transcriptional response they elicit can be at the same time so fast and so precise.

Inheritance of H3K9 methylation regulates genome architecture in Drosophila early embryos

Constitutive heterochromatin is essential for transcriptional silencing and genome integrity. The establishment of constitutive heterochromatin in early embryos and its role in early fruitfly development are unknown. Lysine 9 trimethylation of histone H3 (H3K9me3) and recruitment of its epigenetic reader, heterochromatin protein 1a (HP1a), are hallmarks of constitutive heterochromatin. Here, we show that H3K9me3 is transmitted from the maternal germline to the next generation. Maternally inherited H3K9me3, and the histone methyltransferases (HMT) depositing it, are required for the organization of constitutive heterochromatin: early embryos lacking H3K9 methylation display de-condensation of pericentromeric regions, centromere-centromere de-clustering, mitotic defects, and nuclear shape irregularities, resulting in embryo lethality. Unexpectedly, quantitative CUT&Tag and 4D microscopy measurements of HP1a coupled with biophysical modeling revealed that H3K9me2/3 is largely dispensable for HP1a recruitment. Instead, the main function of H3K9me2/3 at this developmental stage is to drive HP1a clustering and subsequent heterochromatin compaction. Our results show that HP1a binding to constitutive heterochromatin in the absence of H3K9me2/3 is not sufficient to promote proper embryo development and heterochromatin formation. The loss of H3K9 HMTs and H3K9 methylation alters genome organization and hinders embryonic development.

Rapid response of fly populations to gene dosage across development and generations

Although the effects of genetic and environmental perturbations on multicellular organisms are rarely restricted to single phenotypic layers, our current understanding of how developmental programs react to these challenges remains limited. Here, we have examined the phenotypic consequences of disturbing the bicoid regulatory network in early Drosophila embryos. We generated flies with two extra copies of bicoid, which causes a posterior shift of the network’s regulatory outputs and a decrease in fitness. We subjected these flies to EMS mutagenesis, followed by experimental evolution. After only 8–15 generations, experimental populations have normalized patterns of gene expression and increased survival. Using a phenomics approach, we find that populations were normalized through rapid increases in embryo size driven by maternal changes in metabolism and ovariole development. We extend our results to additional populations of flies, demonstrating predictability. Together, our results necessitate a broader view of regulatory network evolution at the systems level.

A phase diagram structure determines the optimal sensitivity-precision trade-off in signaling systems

Signal transduction is crucial for many biological functions. However, it is still unclear how signaling systems function accurately under noisy situations. More specifically, such systems operate in a regime of low response noise, while maintaining high sensitivity to signals. To gain further insight on this regime, here we derive a fundamental trade-off between response sensitivity and precision in biological signaling processes under the static noise condition. We find that the optimal trade-off in signaling networks can be better characterized by a phase diagram structure rather than topological structures. We confirm that the patterning network of early Drosophila embryos agrees with our derived relationship, and apply the optimal phase diagram structure to quantitatively predict the patterning position shifts of the downstream genes, including hunchback, Krüppel, giant, knirps and even-skipped, upon the dosage perturbation of the morphogen Bicoid.

The positioning mechanics of microtubule asters in Drosophila embryo explants.

Microtubule asters are essential in localizing the action of microtubules in processes including mitosis and organelle positioning. In large cells, such as the one-cell sea urchin embryo, aster dynamics are dominated by hydrodynamic pulling forces. However, in systems with more densely positioned nuclei such as the early Drosophila embryo, which packs around 6000 nuclei within the syncytium in a crystalline-like order, it is unclear what processes dominate aster dynamics. Here, we take advantage of a cell cycle regulation Drosophila mutant to generate embryos with multiple asters, independent from nuclei. We use an ex vivo assay to further simplify this biological system to explore the forces generated by and between asters. Through live imaging, drug and optical perturbations, and theoretical modeling, we demonstrate that these asters likely generate an effective pushing force over short distances.

The positioning mechanics of microtubule asters in Drosophila embryo explants

Microtubule asters are essential in localizing the action of microtubules in processes including mitosis and organelle positioning. In large cells, such as the one-cell sea urchin embryo, aster dynamics are dominated by hydrodynamic pulling forces. However, in systems with more densely positioned nuclei such as the early Drosophila embryo, which packs around 6000 nuclei within the syncytium in a crystalline-like order, it is unclear what processes dominate aster dynamics. Here, we take advantage of a cell cycle regulation Drosophila mutant to generate embryos with multiple asters, independent from nuclei. We use an ex vivo assay to further simplify this biological system to explore the forces generated by and between asters. Through live imaging, drug and optical perturbations, and theoretical modeling, we demonstrate that these asters likely generate an effective pushing force over short distances.

Zelda’s Role in Controlling Gene Expression in the Early Embryo

Much research investigates the influence of Zelda (Zinc-finger early Drosophila Embryo activator, Zld) on early embryo development. CAGGTAG and related sequences are common in the early transcribed genes in Drosophila. These are all referred to as sites that can activate transcription and are responsible for maternal RNA degradation (as cited in Liang et al., 2008). However, it is still unknown whether other activators can help gene expression. Here, we analyze the effect of Zld on specific genes, such as Halo, by chromatin immunoprecipitation assays. One result is that the gene expression is low in the absence of Zld, which could suggest that Zld plays an important role in the activation of early development of the Drosophila embryo.

Multifaceted effects on even-skipped transcriptional dynamics upon Krüppel dosage changes.

Although fluctuations in transcription factor (TF) dosage are often well tolerated, TF dosage modulation can change the target gene expression dynamics and result in significant non-lethal developmental phenotypes. Using MS2/MCP-mediated quantitative live imaging in early Drosophila embryos, we analyzed how changing levels of the gap gene Krüppel (Kr) affects transcriptional dynamics of the pair-rule gene even-skipped (eve). Halving the Kr dosage leads to a transient posterior expansion of the eve stripe 2 and an anterior shift of stripe 5. Surprisingly, the most significant changes are observed in eve stripes 3 and 4, the enhancers of which do not contain Kr-binding sites. In Kr heterozygous embryos, both stripes 3 and 4 display narrower widths, anteriorly shifted boundaries and reduced mRNA production levels. We show that Kr dosage indirectly affects stripe 3 and 4 dynamics by modulating other gap gene dynamics. We quantitatively correlate moderate body segment phenotypes of Kr heterozygotes with spatiotemporal changes in eve expression. Our results indicate that nonlinear relationships between TF dosage and phenotypes underlie direct TF-DNA and indirect TF-TF interactions.

Long-range formation of the Bicoid gradient requires multiple dynamic modes that spatially vary across the embryo.

Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution, yet how they form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. Using fluorescence correlation spectroscopy we find there are spatial differences in Bcd diffusivity along the AP axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. In the nucleus, we demonstrate that Bcd dynamics are impacted by binding to DNA. Addition of the Bcd homeodomain to eGFP::NLS qualitatively replicates the Bcd concentration profile, suggesting this domain regulates Bcd dynamics. Our results reveal how a long-range gradient can form while retaining a steep profile through much of its range.

Intrinsic protein disorder is insufficient to drive subnuclear clustering in embryonic transcription factors.

Modern microscopy has revealed that core nuclear functions, including transcription, replication, and heterochromatin formation, occur in spatially restricted clusters. Previous work from our lab has shown that subnuclear high-concentration clusters of transcription factors may play a role in regulating RNA synthesis in the early Drosophila embryo. A nearly ubiquitous feature of eukaryotic transcription factors is that they contain intrinsically disordered regions (IDRs) that often arise from low complexity amino acid sequences within the protein. It has been proposed that IDRs within transcription factors drive co-localization of transcriptional machinery and target genes into high-concentration clusters within nuclei. Here, we test that hypothesis directly, by conducting a broad survey of the subnuclear localization of IDRs derived from transcription factors. Using a novel algorithm to identify IDRs in the Drosophila proteome, we generated a library of IDRs from transcription factors expressed in the early Drosophila embryo. We used this library to perform a high-throughput imaging screen in Drosophila Schneider-2 (S2) cells. We found that while subnuclear clustering does not occur when the majority of IDRs are expressed alone, it is frequently seen in full-length transcription factors. These results are consistent in live Drosophila embryos, suggesting that IDRs are insufficient to drive the subnuclear clustering behavior of transcription factors. Furthermore, the clustering of transcription factors in living embryos was unaffected by the deletion of IDR sequences. Our results demonstrate that IDRs are unlikely to be the primary molecular drivers of the clustering observed during transcription, suggesting a more complex and nuanced role for these disordered protein sequences.

Intrinsic protein disorder is insufficient to drive subnuclear clustering in embryonic transcription factors

Modern microscopy has revealed that core nuclear functions, including transcription, replication, and heterochromatin formation, occur in spatially restricted clusters. Previous work from our lab has shown that subnuclear high-concentration clusters of transcription factors may play a role in regulating RNA synthesis in the early Drosophila embryo. A nearly ubiquitous feature of eukaryotic transcription factors is that they contain intrinsically disordered regions (IDRs) that often arise from low complexity amino acid sequences within the protein. It has been proposed that IDRs within transcription factors drive co-localization of transcriptional machinery and target genes into high-concentration clusters within nuclei. Here, we test that hypothesis directly, by conducting a broad survey of the subnuclear localization of IDRs derived from transcription factors. Using a novel algorithm to identify IDRs in the Drosophila proteome, we generated a library of IDRs from transcription factors expressed in the early Drosophila embryo. We used this library to perform a high-throughput imaging screen in Drosophila Schneider-2 (S2) cells. We found that while subnuclear clustering does not occur when the majority of IDRs are expressed alone, it is frequently seen in full-length transcription factors. These results are consistent in live Drosophila embryos, suggesting that IDRs are insufficient to drive the subnuclear clustering behavior of transcription factors. Furthermore, the clustering of transcription factors in living embryos was unaffected by the deletion of IDR sequences. Our results demonstrate that IDRs are unlikely to be the primary molecular drivers of the clustering observed during transcription, suggesting a more complex and nuanced role for these disordered protein sequences.

Gene co-regulation caused by the special positional relationship in the early stages of Drosophila embryos

The genetic regulation mechanism of early Drosophila embryo has been a research hotspot in the past few decades. Understanding the early genetic regulation mechanisms of Drosophila embryos plays an important role in understanding the growth and development of other animals. In previous studies, an important Transcription Activator, Zelda (Zld), was reported. Zld is a global activator of early embryonic transcription. During the early development of embryos, all animals undergo the material-to-zygotic Transition (MZT), where Zld plays an important role in degrading maternal mRNA and mediating the expression of over 120 zygotic genes. The absence or mutation of Zld will directly lead to the inability of the embryo to complete the MZT process and result in lethality. TAGteam is a special sequence upstream of many genes in Drosophila, and Zld plays a role as a binding protein of TAGteam. TAGteam is a highly conserved sequence that generally exists in the form of CAGGTAG. Mutations or lack of TAGteam often result in inability to bind to Zld, resulting in the inability of genes to be expressed normally. The two genes involved in this experiment, CG14014 (2L: 5549709.. 550835 [-]) and CG18269 (2L: 5551838.. 5552756 [+]), are two adjacent genes on the Drosophila chromosome. They are located on two different DNA strands and facing in opposite directions, while their current role is not yet clear. There are typical TAGteam sequences upstream of both genes. This article conducts experiments to verify the relationship between these two genes and the regulation of Zld protein, and explores the possibility of their co-regulation by designing experiments.

Multi-level regulation of even-skipped stripes by the ubiquitous factor Zelda.

The zinc-finger protein Zelda (Zld) is a key activator of zygotic transcription in early Drosophila embryos. Here, we study Zld-dependent regulation of the seven-striped pattern of the pair-rule gene even-skipped (eve). Individual stripes are regulated by discrete enhancers that respond to broadly distributed activators; stripe boundaries are formed by localized repressors encoded by the gap genes. The strongest effects of Zld are on stripes 2, 3 and 7, which are regulated by two enhancers in a 3.8 kb genomic fragment that includes the eve basal promoter. We show that Zld facilitates binding of the activator Bicoid and the gap repressors to this fragment, consistent with its proposed role as a pioneer protein. To test whether the effects of Zld are direct, we mutated all canonical Zld sites in the 3.8 kb fragment, which reduced expression but failed to phenocopy the abolishment of stripes caused by removing Zld in trans. We show that Zld also indirectly regulates the eve stripes by establishing specific gap gene expression boundaries, which provides the embryonic spacing required for proper stripe activation.

TM2D3, a mammalian homologue of Drosophila neurogenic gene product Almondex, regulates surface presentation of Notch receptors

Notch signaling is an evolutionarily conserved mechanism required for numerous types of cell fate decisions in metazoans. It mediates short-range communication between cells with receptors and ligands, both of which are expressed on the cell surfaces. In response to the ligand-receptor interaction, the ligand and the extracellular domain of the Notch receptor (NECD) in the complex are internalized into ligand-expressing cells by endocytosis, a prerequisite process for the conformational change of the membrane proximal region of Notch to induce critical proteolytic cleavages for its activation. Here we report that overexpression of transmembrane 2 (TM2) domain containing 3 (TM2D3), a mammalian homologue of Drosophila melanogaster Almondex (Amx), activates Notch1. This activation requires the ligand-binding domain in Notch1 and the C-terminal region containing TM2 domain in TM2D3. TM2D3 physically associates with Notch1 at the region distinct from the ligand-binding domain and enhances expression of Notch1 on the cell surface. Furthermore, cell surface expression of Notch1 and Notch2 is reduced in Tm2d3-deficient cells. Finally, amx-deficient Drosophila early embryos exhibit impaired endocytosis of NECD and Delta ligand, for which surface presentation of Notch is required. These results indicate that TM2D3 is an element involved in Notch signaling through the surface presentation.