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.

Body plan evolvability: The role of variability in gene regulatory networks.

Recent computational modeling of early fruit fly (Drosophila) development has characterized the degree to which gene regulation networks can be robust to natural variability. In the first few hours of development, broad spatial gradients of maternally derived transcription factors activate embryonic gap genes. These gap patterns determine the subsequent segmented insect body plan through pair-rule gene expression. Gap genes are expressed with greater spatial precision than the maternal patterns. Computational modeling of the gap-gap regulatory interactions provides a mechanistic understanding for this robustness to maternal variability in wild-type (WT) patterning. A long-standing question in evolutionary biology has been how a system which is robust, such as the developmental program creating any particular species' body plan, is also evolvable, i.e. how can a system evolve or speciate, if the WT form is strongly buffered and protected? In the present work, we use the WT model to explore the breakdown of such Waddington-type 'canalization'. What levels of variability will push the system out of the WT form; are there particular pathways in the gene regulatory mechanism which are more susceptible to losing the WT form; and when robustness is lost, what types of forms are most likely to occur (i.e. what forms lie near the WT)? Manipulating maternal effects in several different pathways, we find a common gap 'peak-to-step' pattern transition in the loss of WT. We discuss these results in terms of the evolvability of insect segmentation, and in terms of experimental perturbations and mutations which could test the model predictions. We conclude by discussing the prospects for using continuum models of pattern dynamics to investigate a wider range of evo-devo problems.

Multi-labeling Live Imaging to Quantify Gene Expression Dynamics During Drosophila Embryonic Development.

Transcription in developing metazoans is inherently stochastic, involving transient and dynamic interactions among transcriptional machinery. A fundamental challenge with traditional techniques, including fixed-tissue protein and RNA staining, is the lack of temporal resolution. Quantifying kinetic changes in transcription can elucidate underlying mechanisms of interaction among regulatory modules. In this protocol, we describe the successful implementation of a combination of MS2/MCP and PP7/PCP systems in living Drosophila embryos to further our understanding of transcriptional dynamics during development. Our technique can be extended to visualize transcriptional activities of multiple genes or alleles simultaneously, characterize allele-specific expression of a target gene, and quantitatively analyze RNA polymerase II activity in a single-cell resolution.

Mechanical Compression of Drosophila Embryos Using Rapid Fabrication Microfluidic Devices.

Microfluidic devices support developmental and mechanobiology studies by enabling the precise control of electrical, chemical, and mechanical stimuli at the microscale. Here, we describe the fabrication of customizable microfluidic devices and demonstrate their efficacy in applying mechanical loads to micro-organs and whole organisms, such as Drosophila embryos. The fabrication technique consists in the use of xurography to define channels and chambers using thin layers of thermoplastics and glass. The superposition of layers followed by thermal lamination produces robust and reproducible devices that are easily adapted for a variety of experiments. The integration of deformable layers and glass in these devices facilitates the imaging of cellular and molecular dynamics in biological specimens under mechanical loads. The method is highly adaptable for studies in mechanobiology.

Par3/bazooka binds NICD and promotes notch signaling during Drosophila development.

The conserved bazooka (baz/par3) gene acts as a key regulator of asymmetrical cell divisions across the animal kingdom. Associated Par3/Baz-Par6-aPKC protein complexes are also well known for their role in the establishment of apical/basal cell polarity in epithelial cells. Here we define a novel, positive function of Baz/Par3 in the Notch pathway. Using Drosophila wing and eye development, we demonstrate that Baz is required for Notch signaling activity and optimal transcriptional activation of Notch target genes. Baz appears to act independently of aPKC in these contexts, as knockdown of aPKC does not cause Notch loss-of-function phenotypes. Using transgenic Notch constructs, our data positions Baz activity downstream of activating Notch cleavage steps and upstream of Su(H)/CSL transcription factor complex activity on Notch target genes. We demonstrate a biochemical interaction between NICD and Baz, suggesting that Baz is required for NICD activity before NICD binds to Su(H). Taken together, our data define a novel role of the polarity protein Baz/Par3, as a positive and direct regulator of Notch signaling through its interaction with NICD.

Confinement promotes nematic alignment of spindle-shaped cells during Drosophila embryogenesis.

Tissue morphogenesis is often controlled by actomyosin networks pulling on adherens junctions (AJs), but junctional myosin levels vary. At an extreme, the Drosophila embryo amnioserosa forms a horseshoe-shaped strip of aligned, spindle-shaped cells lacking junctional myosin. What are the bases of amnioserosal cell interactions and alignment? Compared with surrounding tissue, we find that amnioserosal AJ continuity has lesser dependence on α-catenin, the mediator of AJ-actomyosin association, and greater dependence on Bazooka/Par-3, a junction-associated scaffold protein. Microtubule bundles also run along amnioserosal AJs and support their long-range curvilinearity. Amnioserosal confinement is apparent from partial overlap of its spindle-shaped cells, its outward bulging from surrounding tissue and from compressive stress detected within the amnioserosa. Genetic manipulations that alter amnioserosal confinement by surrounding tissue also result in amnioserosal cells losing alignment and gaining topological defects characteristic of nematically ordered systems. With Bazooka depletion, confinement by surrounding tissue appears to be relatively normal and amnioserosal cells align despite their AJ fragmentation. Overall, the fully elongated amnioserosa appears to form through tissue-autonomous generation of spindle-shaped cells that nematically align in response to confinement by surrounding tissue.

Embryo Microinjection for Transgenesis in Drosophila.

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope. Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

A microRNA that controls the emergence of embryonic movement.

Movement is a key feature of animal systems, yet its embryonic origins are not fully understood. Here, we investigate the genetic basis underlying the embryonic onset of movement in Drosophila focusing on the role played by small non-coding RNAs (microRNAs, miRNAs). To this end, we first develop a quantitative behavioural pipeline capable of tracking embryonic movement in large populations of fly embryos, and using this system, discover that the Drosophila miRNA miR-2b-1 plays a role in the emergence of movement. Through the combination of spectral analysis of embryonic motor patterns, cell sorting and RNA in situs, genetic reconstitution tests, and neural optical imaging we define that miR-2b-1 influences the emergence of embryonic movement by exerting actions in the developing nervous system. Furthermore, through the combination of bioinformatics coupled to genetic manipulation of miRNA expression and phenocopy tests we identify a previously uncharacterised (but evolutionarily conserved) chloride channel encoding gene - which we term Movement Modulator (Motor) - as a genetic target that mechanistically links miR-2b-1 to the onset of movement. Cell-specific genetic reconstitution of miR-2b-1 expression in a null miRNA mutant background, followed by behavioural assays and target gene analyses, suggest that miR-2b-1 affects the emergence of movement through effects in sensory elements of the embryonic circuitry, rather than in the motor domain. Our work thus reports the first miRNA system capable of regulating embryonic movement, suggesting that other miRNAs are likely to play a role in this key developmental process in Drosophila as well as in other species.

Protocol to quantify the rate of RNA polymerase II elongation with MS2/MCP- and PP7/PCP-based live-imaging systems in early Drosophila embryos.

The MS2-PP7 two-color live-imaging system provides insights into the spatiotemporal dynamics of nascent transcripts at tagged loci. Here, we present a protocol to quantitatively measure the rate of RNA polymerase II elongation for each actively transcribing nucleus in living Drosophila embryos. The elongation rate is calculated by measuring the effective distance and the time elapsed between MS2 and PP7 trajectories. We describe steps for preparing embryo samples, performing live imaging, and measuring the elongation rate. For complete details on the use and execution of this protocol, please refer to Keller et al.1.

Two distinct mechanisms of Plexin A function in Drosophila optic lobe lamination and morphogenesis.

Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. We find that, in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In Plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of semaphorin function indicates that Semaphorin 1a, acting in a subset of medulla neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A has little effect on the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of Plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles.

40†years of homeodomain transcription factors in the Drosophila nervous system.

Drosophila nervous system development progresses through a series of well-characterized steps in which homeodomain transcription factors (HDTFs) play key roles during most, if not all, phases. Strikingly, although some HDTFs have only one role, many others are involved in multiple steps of the developmental process. Most Drosophila HDTFs engaged in nervous system development are conserved in vertebrates and often play similar roles during vertebrate development. In this Spotlight, we focus on the role of HDTFs during embryogenesis, where they were first characterized.

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.

The people behind the papers - Jingjing Sun and Angelike Stathopoulos.

Collective migration of caudal visceral mesoderm (CVM) cells in Drosophila embryos helps form the longitudinal muscles of the larval gut. In their study, Angelike Stathopoulos and colleagues reveal that cell division coordinates two gene expression programmes in migrating CVM cells. To know more about their work, we spoke to the first author, Jingjing Sun, and the corresponding author, Angelike Stathopoulos, Professor in the Division of Biology at the California Institute of Technology, USA.

Polarised cell intercalation during Drosophila axis extension is robust to an orthogonal pull by the invaginating mesoderm.

As tissues grow and change shape during animal development, they physically pull and push on each other, and these mechanical interactions can be important for morphogenesis. During Drosophila gastrulation, mesoderm invagination temporally overlaps with the convergence and extension of the ectodermal germband; the latter is caused primarily by Myosin II-driven polarised cell intercalation. Here, we investigate the impact of mesoderm invagination on ectoderm extension, examining possible mechanical and mechanotransductive effects on Myosin II recruitment and polarised cell intercalation. We find that the germband ectoderm is deformed by the mesoderm pulling in the orthogonal direction to germband extension (GBE), showing mechanical coupling between these tissues. However, we do not find a significant change in Myosin II planar polarisation in response to mesoderm invagination, nor in the rate of junction shrinkage leading to neighbour exchange events. We conclude that the main cellular mechanism of axis extension, polarised cell intercalation, is robust to the mesoderm invagination pull. We find, however, that mesoderm invagination slows down the rate of anterior-posterior cell elongation that contributes to axis extension, counteracting the tension from the endoderm invagination, which pulls along the direction of GBE.

Two sequential gene expression programs bridged by cell division support long-distance collective cell migration.

The precise assembly of tissues and organs relies on spatiotemporal regulation of gene expression to coordinate the collective behavior of cells. In Drosophila embryos, the midgut musculature is formed through collective migration of caudal visceral mesoderm (CVM) cells, but how gene expression changes as cells migrate is not well understood. Here, we have focused on ten genes expressed in the CVM and the cis-regulatory sequences controlling their expression. Although some genes are continuously expressed, others are expressed only early or late during migration. Late expression relates to cell cycle progression, as driving string/Cdc25 causes earlier division of CVM cells and accelerates the transition to late gene expression. In particular, we found that the cell cycle effector transcription factor E2F1 is a required input for the late gene CG5080. Furthermore, whereas late genes are broadly expressed in all CVM cells, early gene transcripts are polarized to the anterior or posterior ends of the migrating collective. We show this polarization requires transcription factors Snail, Zfh1 and Dorsocross. Collectively, these results identify two sequential gene expression programs bridged by cell division that support long-distance directional migration of CVM cells.

Kinesin-1 patterns Par-1 and Rho signaling at the cortex of syncytial embryos of Drosophila.

The cell cortex of syncytial Drosophila embryos is patterned into cap and intercap regions by centrosomes, specific sets of proteins that are restricted to their respective regions by unknown mechanisms. Here, we found that Kinesin-1 is required for the restriction of plus- and minus-ends of centrosomal and non-centrosomal microtubules to the cap region, marked by EB1 and Patronin/Shot, respectively. Kinesin-1 also directly or indirectly restricts proteins and Rho signaling to the intercap, including the RhoGEF Pebble, Dia, Myosin II, Capping protein-α, and the polarity protein Par-1. Furthermore, we found that Par-1 is required for cap restriction of Patronin/Shot, and vice versa Patronin, for Par-1 enrichment at the intercap. In summary, our data support a model that Kinesin-1 would mediate the restriction of centrosomal and non-centrosomal microtubules to a region close to the centrosomes and exclude Rho signaling and Par-1. In addition, mutual antagonistic interactions would refine and maintain the boundary between cap and intercap and thus generate a distinct cortical pattern.

Medioapical contractile pulses coordinated between cells regulate Drosophila eye morphogenesis.

Lattice cells (LCs) in the developing Drosophila retina change shape before attaining final form. Previously, we showed that repeated contraction and expansion of apical cell contacts affect these dynamics. Here, we describe another factor, the assembly of a Rho1-dependent medioapical actomyosin ring formed by nodes linked by filaments that contract the apical cell area. Cell area contraction alternates with relaxation, generating pulsatile changes in cell area that exert force on neighboring LCs. Moreover, Rho1 signaling is sensitive to mechanical changes, becoming active when tension decreases and cells expand, while the negative regulator RhoGAP71E accumulates when tension increases and cells contract. This results in cycles of cell area contraction and relaxation that are reciprocally synchronized between adjacent LCs. Thus, mechanically sensitive Rho1 signaling controls pulsatile medioapical actomyosin contraction and coordinates cell behavior across the epithelium. Disrupting the kinetics of pulsing can lead to developmental errors, suggesting this process controls cell shape and tissue integrity during epithelial morphogenesis of the retina.

Effects of N-acetylcysteine on CG8005 gene-mediated proliferation and apoptosis of Drosophila S2 embryonic cells.

To investigate the effect of the antioxidant N-acetylcysteine (NAC) on the proliferation and apoptosis in CG8005 gene-interfering Drosophila S2 embryonic cells by scavenging intracellular reactive oxygen species (ROS). The interfering efficiency of CG8005 gene in Drosophila S2 embryonic cells was verified by real-time quantitative PCR (qRT-PCR). Different concentrations of NAC and phosphate buffered saline (PBS) were used to affect the Drosophila S2 embryonic cells. The growth state of Drosophila S2 embryonic cells was observed by light microscope. Two probes dihydroethidium (DHE) and 2,7-dichlorodihydrofluorescein-acetoacetate (DCFH-DA) were used to observe the ROS production in each group after immunofluorescence staining. TUNEL staining and flow cytometry were used to investigate the apoptosis level of Drosophila S2 embryos, and CCK-8 (Cell Counting Kit-8) was used to detect the cell viability of Drosophila S2 embryos. The knockdown efficiency of siCG8005-2 fragment was high and stable, which was verified by interference efficiency (P < 0.05). There was no significant change in the growth of Drosophila S2 embryonic cells after the treatment of NAC as compared to PBS group. Moreover, knockdowning CG8005 gene resulted in an increase in ROS and apoptosis in Drosophila S2 embryonic cells (P < 0.05) and a decrease in proliferation activity (P < 0.05). In addition, the pretreatment of antioxidant NAC could inhibit ROS production in Drosophila S2 embryonic cells (P < 0.05), reduce cell apoptosis (P < 0.05), and improve cell survival (P < 0.05). The CG8005 gene in Drosophila S2 embryonic cells could regulate the proliferation and apoptosis of S2 embryonic cells by disrupting the redox homeostasis, and antioxidant NAC could inhibit cell apoptosis and promotes cell proliferation by scavenging ROS in Drosophila S2 embryonic cells, which is expected to provide novel insights for the pathogenesis of male infertility and spermatogenesis.

Macrophage Nrf 2 the rescue.

The exuberant phagocytosis of apoptotic cell corpses by macrophages in Drosophila embryos creates highly oxidative environments. Stow and Sweet discuss work from Clemente and Weavers (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202203062) showing for the first time how macrophage Nrf2 is primed to help sustain immune function and mitigate bystander oxidative damage.

The effects of Hh morphogen source movement on signaling dynamics.

Morphogens of the Hh family trigger gene expression changes in receiving cells in a concentration-dependent manner to regulate their identity, proliferation, death or metabolism, depending on the tissue or organ. This variety of responses relies on a conserved signaling pathway. Its logic includes a negative-feedback loop involving the Hh receptor Ptc. Here, using experiments and computational models we study and compare the different spatial signaling profiles downstream of Hh in several developing Drosophila organs. We show that the spatial distributions of Ptc and the activator transcription factor CiA in wing, antenna and ocellus show similar features, but are markedly different from that in the compound eye. We propose that these two profile types represent two time points along the signaling dynamics, and that the interplay between the spatial displacement of the Hh source in the compound eye and the negative-feedback loop maintains the receiving cells effectively in an earlier stage of signaling. These results show how the interaction between spatial and temporal dynamics of signaling and differentiation processes may contribute to the informational versatility of the conserved Hh signaling pathway.