Dynamic Regulatory Processes Research Articles

A novel luciferase-based reporter tool to monitor the dynamics of carbon catabolite repression in filamentous fungi.

Filamentous fungi with their diverse inventory of carbohydrate-active enzymes promise a holistic usage of lignocellulosic residues. A major challenge for application is the inherent repression of enzyme production by carbon catabolite repression (CCR). In the presence of preferred carbon sources, the transcription factor CreA/CRE-1 binds to specific but conserved motifs in promoters of genes involved in sugar metabolism, but the status of CCR is notoriously difficult to quantify. To allow for a real-time evaluation of CreA/CRE-1-mediated CCR at the transcriptional level, we developed a luciferase-based construct, representing a dynamic, highly responsive reporter system that is inhibited by monosaccharides in a quantitative fashion. Using this tool, CreA/CRE-1-dependent CCR triggered by several monosaccharides could be measured in Neurospora crassa, Aspergillus niger and Aspergillus nidulans over the course of hours, demonstrating distinct and dynamic regulatory processes. Furthermore, we used the reporter to visualize the direct impacts of multiple CreA truncations on CCR induction. Our reporter thus offers a widely applicable quantitative approach to evaluate CreA/CRE-1-mediated CCR across diverse fungal species and will help to elucidate the multifaceted effects of CCR on fungal physiology for both basic research and industrial strain engineering endeavours.

Nrf2 Signaling Pathway: Focus on Oxidative Stress in Spinal Cord Injury.

Spinal cord injury (SCI) is a serious, disabling injury to the central nervous system that can lead to motor, sensory, and autonomic dysfunction below the injury plane. SCI can be divided into primary injury and secondary injury according to its pathophysiological process. Primary injury is irreversible in most cases, while secondary injury is a dynamic regulatory process. Secondary injury involves a series of pathological events, such as ischemia, oxidative stress, inflammatory events, apoptotic pathways, and motor dysfunction. Among them, oxidative stress is an important pathological event of secondary injury. Oxidative stress causes a series of destructive events such as lipid peroxidation, DNA damage, inflammation, and cell death, which further worsens the microenvironment of the injured site and leads to neurological dysfunction. The nuclear factor erythrocyte 2-associated factor 2 (Nrf2) is considered to be a key pathway of antioxidative stress and is closely related to the pathological process of SCI. Activation of this pathway can effectively inhibit the oxidative stress process and promote the recovery of nerve function after SCI. Therefore, the Nrf2 pathway may be a potential therapeutic target for SCI. This review deeply analyzed the generation of oxidative stress in SCI, the role and mechanism of Nrf2 as the main regulator of antioxidant stress in SCI, and the influence of cross-talk between Nrf2 and related pathways that may be involved in the pathological regulation of SCI on oxidative stress, and summarized the drugs and other treatment methods based on Nrf2 pathway regulation. The objective of this paper is to provide evidence for the role of Nrf2 activation in SCI and to highlight the important role of Nrf2 in alleviating SCI by elucidating the mechanism, so as to provide a theoretical basis for targeting Nrf2 pathway as a therapy for SCI.

Unraveling the ultrastructure and dynamics of autophagic vesicles: Insights from advanced imaging techniques.

Autophagy, an intracellular self-degradation process, is governed by a complex interplay of signaling pathways and interactions between proteins and organelles. Its fundamental purpose is to efficiently clear and recycle cellular components that are damaged or redundant. Central to this process are autophagic vesicles, specialized structures that encapsulate targeted cellular elements, playing a pivotal role in autophagy. Despite growing interest in the molecular components of autophagic machinery and their regulatory mechanisms,capturing the detailed ultrastructural dynamics of autophagosome formation continues to present significant challenges. However, recent advancements in microscopy, particularly in electron microscopy, have begun to illuminate the dynamic regulatory processes underpinning autophagy. This review endeavors to provide an exhaustive overview of contemporary research on the ultrastructure of autophagic processes. By synthesizing observations from diverse technological methodologies, this review seeks to deepen our understanding of the genesis of autophagic vesicles, their membrane origins, and the dynamic alterations that transpire during the autophagy process. The aim is to bridge gaps in current knowledge and foster a more comprehensive comprehension of this crucial cellular mechanism.

Targeted Therapy of Spinal Cord Injury: Inhibition of Apoptosis Is a Promising Therapeutic Strategy.

Spinal cord injury (SCI) is a serious disabling central nervous system injury that can lead to motor, sensory, and autonomic dysfunction below the injury level. SCI can be divided into primary injury and secondary injury according to pathological process. Primary injury is mostly irreversible, while secondary injury is a dynamic regulatory process. Apoptosis is an important pathological event of secondary injury and has a significant effect on the recovery of nerve function after SCI. Nerve cell death can further aggravate the microenvironment of the injured site, leading to neurological dysfunction and thus affect the clinical outcome of patients. Therefore, apoptosis plays a crucial role in the pathological progression of secondary SCI, while inhibiting apoptosis may be a promising therapeutic strategy for SCI. This review will summarize and explore the factors that lead to cell death after SCI, the influence of cross talk between signaling pathways and pathways involved in apoptosis and discuss the influence of apoptosis on SCI, and the therapeutic significance of targeting apoptosis on SCI. This review helps us to understand the role of apoptosis in secondary SCI and provides a theoretical basis for the treatment of SCI based on apoptosis.

Cleavage of VAMP2/3 Affects Oligodendrocyte Lineage Development in the Developing Mouse Spinal Cord.

In the developing and adult CNS, new oligodendrocytes (OLs) are generated from a population of cells known as oligodendrocyte precursor cells (OPCs). As they begin to differentiate, OPCs undergo a series of highly regulated changes to morphology, gene expression, and membrane organization. This stage represents a critical bottleneck in oligodendrogliogenesis, and the regulatory program that guides it is still not fully understood. Here, we show that in vivo toxin-mediated cleavage of the vesicle associated SNARE proteins VAMP2/3 in the OL lineage of both male and female mice impairs the ability of early OLs to mature into functional, myelinating OLs. In the developing mouse spinal cord, many VAMP2/3-cleaved OLs appeared to stall in the premyelinating, early OL stage, resulting in an overall loss of both myelin density and OL number. The Src kinase Fyn, a key regulator of oligodendrogliogenesis and myelination, is highly expressed among premyelinating OLs, but its expression decreases as OLs mature. We found that OLs with cleaved VAMP2/3 in the spinal cord white matter showed significantly higher expression of Fyn compared with neighboring control cells, potentially because of an extended premyelinating stage. Overall, our results show that functional VAMP2/3 in OL lineage cells is essential for proper myelin formation and plays a major role in controlling the maturation and terminal differentiation of premyelinating OLs.SIGNIFICANCE STATEMENT The production of mature oligodendrocytes (OLs) is essential for CNS myelination during development, myelin remodeling in adulthood, and remyelination following injury or in demyelinating disease. Before myelin sheath formation, newly formed OLs undergo a series of highly regulated changes during a stage of their development known as the premyelinating, or early OL stage. This stage acts as a critical checkpoint in OL development, and much is still unknown about the dynamic regulatory processes involved. In this study, we show that VAMP2/3, SNARE proteins involved in vesicular trafficking and secretion play an essential role in regulating premyelinating OL development and are required for healthy myelination in the developing mouse spinal cord.

Dynamic regulatory processes among child welfare parents: Temporal associations between physiology and parenting behavior.

This study examined how temporal associations between parents' physiological and behavioral responses may reflect underlying regulatory difficulties in at-risk parenting. Time-series data of cardiac indices (second-by-second estimates of inter-beat intervals - IBI, and respiratory sinus arrhythmia - RSA) and parenting behaviors were obtained from 204 child welfare-involved parents (88% mothers, Mage = 32.32 years) during child-led play with their 3- to 7-year-old children (45.1% female; Mage = 4.76 years). Known risk factors for maltreatment, including parents' negative social cognitions, mental health symptoms, and inhibitory control problems, were examined as moderators of intra-individual physiology-behavior associations. Results of ordinary differential equations suggested increases in parents' cardiac arousal at moments when they showed positive parenting behaviors. In turn, higher arousal was associated with momentary decreases in both positive and negative parenting behaviors. Individual differences in these dynamic processes were identified in association with parental risk factors. In contrast, no sample-wide RSA-behavior associations were evident, but a pattern of increased positive parenting at moments of parasympathetic withdrawal emerged among parents showing more total positive parenting behaviors. This study illustrated an innovative and ecologically-valid approach to examining regulatory patterns that may shape parenting in real-time and identified mechanisms that should be addressed in interventions.

Sediments are important in regulating the algae-derived off-flavor (β-cyclocitral) in eutrophic lakes

In recent years, due to global warming and water eutrophication, cyanobacterial blooms have occurred frequently worldwide, resulting in a series of water quality problems, among which the odor problem in lakes is one of the focuses of attention. In the late stage of the bloom, a large amount of algae accumulated on the surface sediment, which will be a great hidden danger to cause odor pollution in lakes. β-Cyclocitral is one of the typical algae-derived odor compounds that cause odor in lakes. In this study, an annual survey of 13 eutrophic lakes in the Taihu Lake basin was investigated to assess the effects of abiotic and biotic factors on β-cyclocitral in water. Our results showed that high concentrations of β-cyclocitral in the pore water (pore-β-cyclocitral) were detected in the sediment and far exceeded that in the water column, with an average of about 100.37 times. Structural equation modeling indicated that algal biomass and pore-β-cyclocitral can directly regulate the concentrations of β-cyclocitral in the water column, and total phosphorus (TP) and temperature (Temp) promoted the algal biomass which further enhanced the production of β-cyclocitral both in the water column and pore water. It was worth noting that when Chla ≥30 μg/L, the effects of algae on pore-β-cyclocitral were significantly enhanced, and pore-β-cyclocitral played a major role in the regulation of β-cyclocitral concentrations in water column. Overall, our study facilitated a comprehensive and systematic understanding of the effects of algae on odorants and the dynamic regulatory processes in complex aquatic ecosystems, and revealed a long-neglected process, that was, the important contribution of sediments to β-cyclocitral in the water column in eutrophic lakes, which would conduce to a more accurate understanding of the evolution of off flavors in lakes and also useful for the management of odors in lakes in the future.

Dynamic Regulatory Processes in the Transition From Suicidal Ideation to Action in Adults Leaving Inpatient Psychiatric Care: Protocol for an Intensive Longitudinal Study.

BackgroundUS suicide rates have risen steadily in the past decade, and suicide risk is especially high in the months after discharge from inpatient psychiatric treatment. However, suicide research has lagged in examining dynamic within-person processes that contribute to risk over time among individuals known to be at high risk of suicide. Almost no research has examined how affective, cognitive, and physiological processes change over minutes, hours, or days to confer risk of suicidal behavior in daily life.ObjectiveThis protocol describes a longitudinal study designed to examine real-world changes in risk of suicide across multiple assessment domains. Specifically, the study involves following adults known to be at high risk of suicide after discharge from inpatient psychiatric care using self-report, interview, actigraphy, and behavioral methods to identify proximal contributors to suicidal thoughts and behaviors. First, we hypothesize that negative affective experiences, which are featured in most major suicide theories, will comprise a latent factor indicative of psychache (emotional pain), which will predict increases in suicidal thinking over time. Second, we hypothesize that poor inhibitory control in the context of negative affective stimuli, as well as emotion-related impulsivity, will predict the transition from suicidal thinking to suicidal behavior over time. Third, we hypothesize that short sleep duration will precede within-person increases in suicidal ideation as well as increased odds of suicidal behavior among those reporting suicidal thoughts.MethodsThe desired sample size is 130 adults with past-week suicidal thoughts or behaviors who are receiving inpatient psychiatric treatment. Participants will complete a battery of measures while on the inpatient unit to assess negative affective experiences, emotion-related impulsivity, inhibitory control, typical sleep patterns, and relevant covariates. After discharge from inpatient care, participants will complete 4 weeks of signal-contingent ecological momentary assessment surveys, as well as mobile behavioral measures of inhibitory control, while wearing an actigraphy device that will gather objective data on sleep. Participants will complete interviews regarding suicidal thoughts and behaviors at 4 and 8 weeks after discharge.ResultsThe study was funded by the National Institutes of Health in November 2020. Recruitment began in April 2021. Data analysis will begin after completion of data collection.ConclusionsThis study will elucidate how affective, cognitive, and physiological risk factors contribute (or do not contribute) to within-person fluctuations in suicide risk in daily life, with important implications for extant theories of suicide. Of import, the examined risk factors are all modifiable; thus, the results will inform identification of key targets for just-in-time, flexible, personalized, digital interventions that can be used to decrease emotional distress and prevent suicide among those at highest risk.International Registered Report Identifier (IRRID)DERR1-10.2196/38582

Host B cells escape CAR-T immunotherapy by reversible downregulation of CD19.

Anti-CD19-CAR-T cells are a successful clinical immunotherapy for B cell lymphomas, although some lymphomas can escape attack by downregulating surface CD19 levels. An undesirable consequence of this therapy is that it can also eliminate healthy B cells expressing CD19. Therefore, understanding the dynamics of CD19 expression in B cells under CAR-T cell immunotherapy can help mitigate both escape and adverse outcomes. Previous studies suggested that mechanisms responsible for the loss of CD19 expression in lymphomas usually involves genomic deletion or epigenetic modification which permanently removes CD19 as a therapeutic target in these cells. We examined if healthy B cells can use similar processes to lose CD19 expression and escape CAR-T attack. In the presence of CAR-T cells, untransformed B cells both when cultured in vitro or in vivo in non-tumor bearing animals downregulate expression of CD19. We then used adoptive transfer strategies to remove CD19-low B cells from αCD19-CAR-T pressure in vivo. Intriguingly, these B cells systematically recovered surface expression of CD19 comparable to wild-type levels. These data suggest that unlike many cases of lymphomas, healthy B cells downregulate CD19 in a reversible fashion. Taken together, these data suggest a dynamic regulatory process of CD19 surface expression on healthy B cells that could be exploited to modulate the expression of CD19 on cancer cells to improve immunotherapy or minimize the depletion of endogenous B cell compartment during treatment.

Human embryoid bodies as a novel system for genomic studies of functionally diverse cell types.

Practically all studies of gene expression in humans to date have been performed in a relatively small number of adult tissues. Gene regulation is highly dynamic and context-dependent. In order to better understand the connection between gene regulation and complex phenotypes, including disease, we need to be able to study gene expression in more cell types, tissues, and states that are relevant to human phenotypes. In particular, we need to characterize gene expression in early development cell types, as mutations that affect developmental processes may be of particular relevance to complex traits. To address this challenge, we propose to use embryoid bodies (EBs), which are organoids that contain a multitude of cell types in dynamic states. EBs provide a system in which one can study dynamic regulatory processes at an unprecedentedly high resolution. To explore the utility of EBs, we systematically explored cellular and gene expression heterogeneity in EBs from multiple individuals. We characterized the various cell types that arise from EBs, the extent to which they recapitulate gene expression in vivo, and the relative contribution of technical and biological factors to variability in gene expression, cell composition, and differentiation efficiency. Our results highlight the utility of EBs as a new model system for mapping dynamic inter-individual regulatory differences in a large variety of cell types.

Dynamic Interplay between Modes of Regulation During Motivationally Challenging Episodes in Collaboration

The cognitive and social demands of collaboration can raise significant motivation challenges. Task progression relies on team members strategically taking control of the problems and adapting accordingly. Theory indicates that productive collaboration involves groups using three modes of regulation: self-regulation, co-regulation, and socially shared regulation. Despite research demonstrating the occurrence of all three modes in collaboration, it is unclear how these modes interact and how co-regulation supports the emergence of self- and shared-regulation of motivation. The study aimed to examine the role co-regulation played in dynamically stimulating the emergence of self- and shared-regulation of motivation. A cross-case comparison was conducted between two groups who experienced high levels of motivation challenges but achieved contrasting perceptions of the overall team learning productivity. During analysis, groups’ dynamic regulatory processes within the online environment were visually represented using a tool called the Chronologically-ordered Representation for Tool-Related Activity (CORDTRA). Findings demonstrate that co-regulation of motivation may afford and thwart the emergence of self- and shared-regulation, and these processes interacted with the group’s situational challenges and the regulatory skills group members possessed. Comparisons between the two groups indicated that groups' motivation regulation should (a) match the demands of the challenges at hand, (b) be positively supported by group members through co-regulation, and (b) involve a more varied strategic responses so that the group may continue to learn and co-construct knowledge effectively as a team.

Typologies of dyadic mother-infant emotion regulation following immunization

Mother-infant dyadic emotion regulation – the joint modulation of affective rhythms as interactive partners dynamically respond to each other across time – has been shown to promote social-emotional wellbeing both during and beyond infancy. Although contributions of dyadic regulation to self-regulatory development may particularly apparent during infant distress, studies have traditionally examined dyadic regulation in low-stress contexts. The present study addresses this gap by identifying distinct patterns of mother-infant dyadic emotion regulation following a highly distressing immunization procedure and then examining how these groups differed in mother and infant personality and temperament characteristics. Mother-infant dyads (N = 131) were videotaped during a routine immunization procedure, and infant crying and maternal soothing behaviors were subsequently coded. Cluster analysis was applied to trajectories of latent states representing each dyad’s post-immunization behaviors. Results indicated five typologies of dyadic regulation following infant immunization. These typologies reflected the effectiveness with which the dyad worked together to soothe infant distress, as well as the specific maternal soothing behaviors employed. Differences in maternal personality and infant temperament among clusters indicated that both mothers and infants contributed to the dynamic regulatory process.

Proteomic Analyses of the Eukaryotic Replication Machinery.

DNA replication in a human cell involves hundreds of proteins that copy the DNA accurately and completely each cell division cycle. In addition to the core DNA copying machine (the replisome), accessory proteins work to respond to replication stress, correct errors, and repackage the DNA into appropriate chromatin structures. New proteomic tools have been invented in the past few years to facilitate the purification, identification, and quantification of the replication, chromatin maturation, and replication stress response machineries. These tools, including iPOND (isolation of proteins on nascent DNA) and NCC (nascent chromatin capture), have yielded discoveries of new proteins involved in these processes and insights into the dynamic regulatory processes ensuring genome and chromatin integrity. In this review, I will introduce these experimental approaches and examine how they have been utilized to define the replication fork proteome.

Astrocytic gap junction blockade markedly increases extracellular potassium without causing seizures in the mouse neocortex

Extracellular potassium concentration, [K+]o, is a major determinant of neuronal excitability. In the healthy brain, [K+]o levels are tightly controlled. During seizures, [K+]o increases up to 15mM and is thought to cause seizures due to its depolarizing effect. Although astrocytes have been suggested to play a key role in the redistribution (or spatial buffering) of excess K+ through Connexin-43 (Cx43)-based Gap Junctions (GJs), the relation between this dynamic regulatory process and seizure generation remains unknown. Here we contrasted the role of astrocytic GJs and hemichannels by studying the effect of GJ and hemichannel blockers on [K+]o regulation in vivo. [K+]o was measured by K+-sensitive microelectrodes. Neuronal excitability was estimated by local field potential (LFP) responses to forepaw stimulation and changes in the power of resting state activity. Starting at the baseline [K+]o level of 1.61±0.3mM, cortical microinjection of CBX, a broad spectrum connexin channel blocker, increased [K+]o to 11±3mM, Cx43 GJ/hemichannel blocker Gap27 increased it from 1.9±0.7 to 9±1mM. At these [K+]o levels, no seizures were observed. Cx43 hemichannel blockade with TAT-Gap19 increased [K+]o by only ~1mM. Microinjection of 4-aminopyridine, a known convulsant, increased [K+]o to ~10mM and induced spontaneously recurring seizures, whereas direct application of K+ did not trigger seizure activity. These findings are the first in vivo demonstration that astrocytic GJs are major determinants for the spatial buffering of [K+]o and that an increase in [K+]o alone does not trigger seizures in the neocortex.

Computational phosphoproteomics: from identification to localization.

Analysis of the phosphoproteome by MS has become a key technology for the characterization of dynamic regulatory processes in the cell, since kinase and phosphatase action underlie many major biological functions. However, the addition of a phosphate group to a suitable side chain often confounds informatic analysis by generating product ion spectra that are more difficult to interpret (and consequently identify) relative to unmodified peptides. Collectively, these challenges have motivated bioinformaticians to create novel software tools and pipelines to assist in the identification of phosphopeptides in proteomic mixtures, and help pinpoint or “localize” the most likely site of modification in cases where there is ambiguity. Here we review the challenges to be met and the informatics solutions available to address them for phosphoproteomic analysis, as well as highlighting the difficulties associated with using them and the implications for data standards.

The heat shock response: A case study of chromatin dynamics in gene regulation

Recent studies in transcriptional regulation using the Drosophila heat shock response system have elucidated many of the dynamic regulatory processes that govern transcriptional activation and repression. The classic view that the control of gene expression occurs at the point of RNA polymerase II (Pol II) recruitment is now giving way to a more complex outlook of gene regulation. Promoter chromatin dynamics coordinate with transcription factor binding to maintain the promoters of active genes accessible. For a large number of genes, the rate-limiting step in Pol II progression occurs during its initial elongation, where Pol II transcribes 30-50 bp and pauses for further signals. These paused genes have unique genic chromatin architecture and dynamics compared with genes where Pol II recruitment is rate limiting for expression. Further elongation of Pol II along the gene causes nucleosome turnover, a continuous process of eviction and replacement, which suggests a potential mechanism for Pol II transit along a nucleosomal template. In this review, we highlight recent insights into transcription regulation of the heat shock response and discuss how the dynamic regulatory processes involved at each transcriptional stage help to generate faithful yet highly responsive gene expression.

Local phosphocycling mediated by LOK/SLK restricts ezrin function to the apical aspect of epithelial cells

In this paper, we describe how a dynamic regulatory process is necessary to restrict microvilli to the apical aspect of polarized epithelial cells. We found that local phosphocycling regulation of ezrin, a critical plasma membrane-cytoskeletal linker of microvilli, was required to restrict its function to the apical membrane. Proteomic approaches and ribonucleic acid interference knockdown identified lymphocyte-oriented kinase (LOK) and SLK as the relevant kinases. Using drug-resistant LOK and SLK variants showed that these kinases were sufficient to restrict ezrin function to the apical domain. Both kinases were enriched in microvilli and locally activated there. Unregulated kinase activity caused ezrin mislocalization toward the basolateral domain, whereas expression of the kinase regulatory regions of LOK or SLK resulted in local inhibition of ezrin phosphorylation by the endogenous kinases. Thus, the domain-specific presence of microvilli is a dynamic process requiring a localized kinase driving the phosphocycling of ezrin to continually bias its function to the apical membrane.

Modeling Dynamic Regulatory Processes in Stroke

The ability to examine the behavior of biological systems in silico has the potential to greatly accelerate the pace of discovery in diseases, such as stroke, where in vivo analysis is time intensive and costly. In this paper we describe an approach for in silico examination of responses of the blood transcriptome to neuroprotective agents and subsequent stroke through the development of dynamic models of the regulatory processes observed in the experimental gene expression data. First, we identified functional gene clusters from these data. Next, we derived ordinary differential equations (ODEs) from the data relating these functional clusters to each other in terms of their regulatory influence on one another. Dynamic models were developed by coupling these ODEs into a model that simulates the expression of regulated functional clusters. By changing the magnitude of gene expression in the initial input state it was possible to assess the behavior of the networks through time under varying conditions since the dynamic model only requires an initial starting state, and does not require measurement of regulatory influences at each time point in order to make accurate predictions. We discuss the implications of our models on neuroprotection in stroke, explore the limitations of the approach, and report that an optimized dynamic model can provide accurate predictions of overall system behavior under several different neuroprotective paradigms.

Multiphoton Imaging of Renal Regulatory Mechanisms

Most physiological functions of the kidneys, including the clearance of metabolic waste products, maintenance of body fluid, electrolyte homeostasis, and blood pressure, are achieved by complex interactions between multiple renal cell types and previously inaccessible structures in many organ parts that have been difficult to study. Multiphoton fluorescence microscopy offers a state-of-the-art imaging technique for deep optical sectioning of living tissues and organs with minimal deleterious effects. Dynamic regulatory processes and multiple functions in the intact kidney can be quantitatively visualized in real time, noninvasively, and with submicron resolution. This article reviews innovative multiphoton imaging technologies and their applications that provided the most complex, immediate, and dynamic portrayal of renal function-clearly depicting as well as analyzing the components and mechanisms involved in renal (patho)physiology.

On the dynamic nature of positional information

Morphogenetic fields are among the most fundamental concepts of embryology. However, they are also among the most ill-defined, since they consist of dynamic regulatory processes whose exact nature remains elusive. In order to achieve a more rigorous definition of a developmental field, Lewis Wolpert introduced the concept of positional information illustrated by his French Flag model. Here we argue that Wolpert's positional information - a static coordinate system defining a field - lacks essential properties of the original field concept. We show how data-driven mathematical modeling approaches now enable us to study regulatory processes in a way that is qualitatively different from our previous level of understanding. As an example, we review our recent analysis of segmentation gene expression in the blastoderm embryo of the fruit fly Drosophila melanogaster. Based on this analysis, we propose a revised French Flag, which incorporates the dynamic, feedback-driven nature of pattern formation in the Drosophila blastoderm.