RESUMO
Deneke et al. (2019) discover that dynamic interactions of cell cycle and actomyosin contractility systems synchronize nuclear cleavages, generating a cytoplasmic flow that results in a spatially uniform distribution of zygotic nuclei in the early Drosophila embryo. This work underscores the importance of self-organizing mechanisms before the onset of zygotic transcription.
Assuntos
Drosophila melanogaster , Drosophila , Animais , Ciclo Celular , Física , ZigotoRESUMO
Large cells often rely on cytoplasmic flows for intracellular transport, maintaining homeostasis, and positioning cellular components. Understanding the mechanisms of these flows is essential for gaining insights into cell function, developmental processes, and evolutionary adaptability. Here, we focus on a class of self-organized cytoplasmic stirring mechanisms that result from fluid-structure interactions between cytoskeletal elements at the cell cortex. Drawing inspiration from streaming flows in late-stage fruit fly oocytes, we propose an analytically tractable active carpet theory. This model deciphers the origins and three-dimensional spatiotemporal organization of such flows. Through a combination of simulations and weakly nonlinear theory, we establish the pathway of the streaming flow to its global attractor: a cell-spanning vortical twister. Our study reveals the inherent symmetries of this emergent flow, its low-dimensional structure, and illustrates how complex fluid-structure interaction aligns with classical solutions in Stokes flow. This framework can be easily adapted to elucidate a broad spectrum of self-organized, cortex-driven intracellular flows.
Assuntos
Citoplasma , Citoesqueleto , Animais , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Modelos Biológicos , Oócitos/metabolismo , Corrente Citoplasmática/fisiologiaRESUMO
When a founder cell and its progeny divide with incomplete cytokinesis, a network forms in which each intercellular bridge corresponds to a past mitotic event. Such networks are required for gamete production in many animals, and different species have evolved diverse final network topologies. Although mechanisms regulating network assembly have been identified in particular organisms, we lack a quantitative framework to understand network assembly and inter-species variability. Motivated by cell networks responsible for oocyte production in invertebrates, where the final topology is typically invariant within each species, we devised a mathematical model for generating cell networks, in which each node is an oscillator and, after a full cycle, the node produces a daughter to which it remains connected. These cell cycle oscillations are transient and coupled via diffusion over the edges of the network. By variation of three biologically motivated parameters, our model generates nearly all such networks currently reported across invertebrates. Furthermore, small parameter variations can rationalize cases of intra-species variation. Because cell networks outside of the ovary often form less deterministically, we propose model generalizations to account for sources of stochasticity.
Assuntos
Citocinese , Modelos Biológicos , Animais , Ciclo Celular , Divisão CelularRESUMO
Positional information in development often manifests as stripes of gene expression, but how stripes form remains incompletely understood. Here, we use optogenetics and live-cell biosensors to investigate the posterior brachyenteron (byn) stripe in early Drosophila embryos. This stripe depends on interpretation of an upstream ERK activity gradient and the expression of two target genes, tailless (tll) and huckebein (hkb), that exert antagonistic control over byn. We find that high or low doses of ERK signaling produce transient or sustained byn expression, respectively. Although tll transcription is always rapidly induced, hkb converts graded ERK inputs into a variable time delay. Nuclei thus interpret ERK amplitude through the relative timing of tll and hkb transcription. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop, which is sufficient to explain byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that 'blurring' of an all-or-none stimulus through intracellular diffusion non-locally produces a byn stripe. Overall, we provide a blueprint for using optogenetics to dissect developmental signal interpretation in space and time.
Assuntos
Núcleo Celular , Drosophila , Animais , Difusão , Embrião de Mamíferos , OptogenéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Loss of normal tissue architecture is a hallmark of oncogenic transformation1. In developing organisms, tissues architectures are sculpted by mechanical forces during morphogenesis2. However, the origins and consequences of tissue architecture during tumorigenesis remain elusive. In skin, premalignant basal cell carcinomas form 'buds', while invasive squamous cell carcinomas initiate as 'folds'. Here, using computational modelling, genetic manipulations and biophysical measurements, we identify the biophysical underpinnings and biological consequences of these tumour architectures. Cell proliferation and actomyosin contractility dominate tissue architectures in monolayer, but not multilayer, epithelia. In stratified epidermis, meanwhile, softening and enhanced remodelling of the basement membrane promote tumour budding, while stiffening of the basement membrane promotes folding. Additional key forces stem from the stratification and differentiation of progenitor cells. Tumour-specific suprabasal stiffness gradients are generated as oncogenic lesions progress towards malignancy, which we computationally predict will alter extensile tensions on the tumour basement membrane. The pathophysiologic ramifications of this prediction are profound. Genetically decreasing the stiffness of basement membranes increases membrane tensions in silico and potentiates the progression of invasive squamous cell carcinomas in vivo. Our findings suggest that mechanical forces-exerted from above and below progenitors of multilayered epithelia-function to shape premalignant tumour architectures and influence tumour progression.
Assuntos
Membrana Basal/metabolismo , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Actomiosina/metabolismo , Animais , Carcinogênese , Proliferação de Células , Simulação por Computador , Progressão da Doença , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Camundongos , Invasividade Neoplásica , MaleabilidadeRESUMO
The decision to stop growing and mature into an adult is a critical point in development that determines adult body size, impacting multiple aspects of an adult's biology. In many animals, growth cessation is a consequence of hormone release that appears to be tied to the attainment of a particular body size or condition. Nevertheless, the size-sensing mechanism animals use to initiate hormone synthesis is poorly understood. Here, we develop a simple mathematical model of growth cessation in Drosophila melanogaster, which is ostensibly triggered by the attainment of a critical weight (CW) early in the last instar. Attainment of CW is correlated with the synthesis of the steroid hormone ecdysone, which causes a larva to stop growing, pupate, and metamorphose into the adult form. Our model suggests that, contrary to expectation, the size-sensing mechanism that initiates metamorphosis occurs before the larva reaches CW; that is, the critical-weight phenomenon is a downstream consequence of an earlier size-dependent developmental decision, not a decision point itself. Further, this size-sensing mechanism does not require a direct assessment of body size but emerges from the interactions between body size, ecdysone, and nutritional signaling. Because many aspects of our model are evolutionarily conserved among all animals, the model may provide a general framework for understanding how animals commit to maturing from their juvenile to adult form.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila melanogaster , Ecdisona , Tamanho Corporal , Larva , Metamorfose BiológicaRESUMO
Mutations in signal transduction pathways lead to various diseases including cancers. MEK1 kinase, encoded by the human MAP2K1 gene, is one of the central components of the MAPK pathway and more than a hundred somatic mutations in the MAP2K1 gene were identified in various tumors. Germline mutations deregulating MEK1 also lead to congenital abnormalities, such as the cardiofaciocutaneous syndrome and arteriovenous malformation. Evaluating variants associated with a disease is a challenge, and computational genomic approaches aid in this process. Establishing evolutionary history of a gene improves computational prediction of disease-causing mutations; however, the evolutionary history of MEK1 is not well understood. Here, by revealing a precise evolutionary history of MEK1, we construct a well-defined dataset of MEK1 metazoan orthologs, which provides sufficient depth to distinguish between conserved and variable amino acid positions. We matched known and predicted disease-causing and benign mutations to evolutionary changes observed in corresponding amino acid positions and found that all known and many suspected disease-causing mutations are evolutionarily intolerable. We selected several variants that cannot be unambiguously assessed by automated prediction tools but that are confidently identified as "damaging" by our approach, for experimental validation in Drosophila. In all cases, evolutionary intolerant variants caused increased mortality and severe defects in fruit fly embryos confirming their damaging nature. We anticipate that our analysis will serve as a blueprint to help evaluate known and novel missense variants in MEK1 and that our approach will contribute to improving automated tools for disease-associated variant interpretation.
Assuntos
Displasia Ectodérmica , Cardiopatias Congênitas , Humanos , Animais , Mutação , Displasia Ectodérmica/genética , Mutação de Sentido Incorreto , Cardiopatias Congênitas/genética , Aminoácidos/genética , MAP Quinase Quinase 1/genéticaRESUMO
Phase separation underlies the organization of the nucleus, including the biogenesis of nucleoli and the packaging of heterochromatin. Here we explore the regulation of transcription factor condensates involved in gene repression by ERK signaling in gastrulating embryos of a simple proto-vertebrate (Ciona). ERK signaling induces nuclear export of the transcriptional repressor Ets-2 repressive factor (ERF), which has been linked to various human developmental disorders. Using high-resolution imaging, we show that ERF is localized within discrete nuclear condensates that dissolve upon ERK activation. Interestingly, we observe dynamic pulses of assembly and dissociation during interphase, providing visualization of a nuclear phase separation process regulated by cell signaling. We discuss the implications of these observations for producing sharp on/off switches in gene activity and suppressing noise in cell-cell signaling events.
Assuntos
Ciona/embriologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Animais , HumanosRESUMO
During early Drosophila embryogenesis, a network of gene regulatory interactions orchestrates terminal patterning, playing a critical role in the subsequent formation of the gut. We utilized CRISPR gene editing at endogenous loci to create live reporters of transcription and light-sheet microscopy to monitor the individual components of the posterior gut patterning network across 90 min prior to gastrulation. We developed a computational approach for fusing imaging datasets of the individual components into a common multivariable trajectory. Data fusion revealed low intrinsic dimensionality of posterior patterning and cell fate specification in wild-type embryos. The simple structure that we uncovered allowed us to construct a model of interactions within the posterior patterning regulatory network and make testable predictions about its dynamics at the protein level. The presented data fusion strategy is a step toward establishing a unified framework that would explore how stochastic spatiotemporal signals give rise to highly reproducible morphogenetic outcomes.
Assuntos
Padronização Corporal , Proteínas de Drosophila , Drosophila melanogaster , Endoderma , Redes Reguladoras de Genes , Animais , Padronização Corporal/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Endoderma/crescimento & desenvolvimento , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
The EGF signaling pathway specifies neuronal identities in the Drosophila embryo by regulating developmental patterning genes such as intermediate neuroblasts defective (ind). EGFR is activated in the ventral midline and neurogenic ectoderm by the Spitz ligand, which is processed by the Rhomboid protease. CRISPR/Cas9 was used to delete defined rhomboid enhancers mediating expression at each site of Spitz processing. Surprisingly, the neurogenic ectoderm, not the ventral midline, was found to be the dominant source of EGF patterning activity. We suggest that Drosophila is undergoing an evolutionary transition in central nervous system (CNS)-organizing activity from the ventral midline to the neurogenic ectoderm.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Embrião não Mamífero/metabolismo , Fator de Crescimento Epidérmico/genética , Receptores ErbB/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Proteínas de Membrana/genética , Neurogênese/genética , Receptores de Peptídeos de Invertebrados/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem da Célula , Células Cultivadas , Sistema Nervoso Central , Drosophila/embriologia , Proteínas de Drosophila/antagonistas & inibidores , Embrião não Mamífero/citologia , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Feminino , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Receptores de Peptídeos de Invertebrados/genética , Transdução de SinaisRESUMO
The extracellular signal-regulated kinase (ERK) controls multiple critical processes in the cell and is deregulated in human cancers, congenital abnormalities, immune diseases, and neurodevelopmental syndromes. Catalytic activity of ERK requires dual phosphorylation by an upstream kinase, in a mechanism that can be described by two sequential Michaelis-Menten steps. The estimation of individual reaction rate constants from kinetic data in the full mechanism has proved challenging. Here, we present an analytically tractable approach to parameter estimation that is based on the phase plane representation of ERK activation and yields two combinations of six reaction rate constants in the detailed mechanism. These combinations correspond to the ratio of the specificities of two consecutive phosphorylations and the probability that monophosphorylated substrate does not dissociate from the enzyme before the second phosphorylation. The presented approach offers a language for comparing the effects of mutations that disrupt ERK activation and function in vivo. As an illustration, we use phase plane representation to analyze dual phosphorylation under heterozygous conditions, when two enzyme variants compete for the same substrate.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular , Humanos , MAP Quinases Reguladas por Sinal Extracelular/química , FosforilaçãoRESUMO
Size is a fundamental feature of living entities and is intimately tied to their function. Scaling laws, which can be traced to D'Arcy Thompson and Julian Huxley, have emerged as a powerful tool for studying regulation of the growth dynamics of organisms and their constituent parts. Yet, throughout the 20th century, as scaling laws were established for single cells, quantitative studies of the coordinated growth of multicellular structures have lagged, largely owing to technical challenges associated with imaging and image processing. Here, we present a supervised learning approach for quantifying the growth dynamics of germline cysts during oogenesis. Our analysis uncovers growth patterns induced by the groupwise developmental dynamics among connected cells, and differential growth rates of their organelles. We also identify inter-organelle volumetric scaling laws, finding that nurse cell growth is linear over several orders of magnitude. Our approach leverages the ever-increasing quantity and quality of imaging data, and is readily amenable for studies of collective cell growth in other developmental contexts, including early mammalian embryogenesis and germline development.
Assuntos
Proliferação de Células/fisiologia , Animais , Evolução Biológica , Biologia do Desenvolvimento/métodos , Dípteros/fisiologia , Células Germinativas/fisiologia , Oogênese/fisiologia , Organelas/fisiologiaRESUMO
From insects to mammals, oocytes and sperm develop within germline cysts comprising cells connected by intercellular bridges (ICBs). In numerous insects, formation of the cyst is accompanied by growth of the fusome-a membranous organelle that permeates the cyst. Fusome composition and function are best understood in Drosophila melanogaster: during oogenesis, the fusome dictates cyst topology and size and facilitates oocyte selection, while during spermatogenesis, the fusome synchronizes the cyst's response to DNA damage. Despite its distinct and sex-specific roles during insect gametogenesis, elucidating fusome growth and inheritance in females and its structure and connectivity in males has remained challenging. Here, we take advantage of advances in three-dimensional (3D) confocal microscopy and computational image processing tools to reconstruct the topology, growth, and distribution of the fusome in both sexes. In females, our experimental findings inform a theoretical model for fusome assembly and inheritance and suggest that oocyte selection proceeds through an 'equivalency with a bias' mechanism. In males, we find that cell divisions can deviate from the maximally branched pattern observed in females, leading to greater topological variability. Our work consolidates existing disjointed experimental observations and contributes a readily generalizable computational approach for quantitative studies of gametogenesis within and across species.
Assuntos
Drosophila melanogaster , Sêmen , Animais , Feminino , Masculino , Drosophila melanogaster/genética , Oócitos , Oogênese/genética , Divisão Celular , MamíferosRESUMO
Markers for the endoderm and mesoderm germ layers are commonly expressed together in the early embryo, potentially reflecting cells' ability to explore potential fates before fully committing. It remains unclear when commitment to a single-germ layer is reached and how it is impacted by external signals. Here, we address this important question in Drosophila, a convenient model system in which mesodermal and endodermal fates are associated with distinct cellular movements during gastrulation. Systematically applying endoderm-inducing extracellular signal-regulated kinase (ERK) signals to the ventral medial embryo-which normally only receives a mesoderm-inducing cue-reveals a critical time window during which mesodermal cell movements and gene expression are suppressed by proendoderm signaling. We identify the ERK target gene huckebein (hkb) as the main cause of the ventral furrow suppression and use computational modeling to show that Hkb repression of the mesoderm-associated gene snail is sufficient to account for a broad range of transcriptional and morphogenetic effects. Our approach, pairing precise signaling perturbations with observation of transcriptional dynamics and cell movements, provides a general framework for dissecting the complexities of combinatorial tissue patterning.
Assuntos
Gástrula/metabolismo , Gastrulação/fisiologia , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Animais , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Endoderma/citologia , Endoderma/embriologia , Gástrula/citologia , Mesoderma/citologia , Mesoderma/embriologiaRESUMO
New studies of metabolic reactions and networks in embryos are making important additions to regulatory models of development, so far dominated by genes and signals. Metabolic control of development is not a new idea and can be traced back to Joseph Needham's 'Chemical Embryology', published in the 1930s. Even though Needham's ideas fell by the wayside with the advent of genetic studies of embryogenesis, they demonstrated that embryos provide convenient models for addressing fundamental questions in biochemistry and are now experiencing a comeback, enabled by the powerful merger of detailed mechanistic studies and systems-level techniques. Here we review recent results from studies that quantified the energy budget of embryogenesis in Drosophila and started to untangle the intricate connections between core anabolic processes and developmental transitions. Dynamic coordination of metabolic, genetic, and signaling networks appears to be essential for seamless progression of development.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/genética , Embrião não Mamífero/citologia , Metabolismo Energético , Redes e Vias Metabólicas , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Embrião não Mamífero/metabolismoRESUMO
In insects, brain-derived Prothoracicotropic hormone (PTTH) activates the receptor tyrosine kinase (RTK) Torso to initiate metamorphosis through the release of ecdysone. We have determined the crystal structure of silkworm PTTH in complex with the ligand-binding region of Torso. Here we show that ligand-induced Torso dimerization results from the sequential and negatively cooperative formation of asymmetric heterotetramers. Mathematical modeling of receptor activation based upon our biophysical studies shows that ligand pulses are "buffered" at low receptor levels, leading to a sustained signal. By contrast, high levels of Torso develop the signal intensity and duration of a noncooperative system. We propose that this may allow Torso to coordinate widely different functions from a single ligand by tuning receptor levels. Phylogenic analysis indicates that Torso is found outside arthropods, including human parasitic roundworms. Together, our findings provide mechanistic insight into how this receptor system, with roles in embryonic and adult development, is regulated.
Assuntos
Bombyx/metabolismo , Hormônios de Inseto/química , Hormônios de Inseto/metabolismo , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Sítios de Ligação , Bombyx/química , Cristalografia por Raios X , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Modelos Moleculares , Filogenia , Multimerização Proteica , Receptores de Interleucina-17/química , Transdução de SinaisRESUMO
Numerous engineered and natural systems form through reinforcement and stabilization of a deformed configuration that was generated by a transient force. An important class of such structures arises during gametogenesis, when a dividing cell undergoes incomplete cytokinesis, giving rise to daughter cells that remain connected through a stabilized intercellular bridge (ICB). ICBs can form through arrest of the contractile cytokinetic furrow and its subsequent stabilization. Despite knowledge of the molecular components, the mechanics underlying robust ICB assembly and the interplay between ring contractility and stiffening are poorly understood. Here, we report joint experimental and theoretical work that explores the physics underlying robust ICB assembly. We develop a continuum mechanics model that reveals the minimal requirements for the formation of stable ICBs, and validate the model's equilibrium predictions through a tabletop experimental analog. With insight into the equilibrium states, we turn to the dynamics: we demonstrate that contractility and stiffening are in dynamic competition and that the time intervals of their action must overlap to ensure assembly of ICBs of biologically observed proportions. Our results highlight a mechanism in which deformation and remodeling are tightly coordinated-one that is applicable to several mechanics-based applications and is a common theme in biological systems spanning several length scales.
Assuntos
CitocineseRESUMO
The famous Arrhenius equation is well suited to describing the temperature dependence of chemical reactions but has also been used for complicated biological processes. Here, we evaluate how well the simple Arrhenius equation predicts complex multi-step biological processes, using frog and fruit fly embryogenesis as two canonical models. We find that the Arrhenius equation provides a good approximation for the temperature dependence of embryogenesis, even though individual developmental intervals scale differently with temperature. At low and high temperatures, however, we observed significant departures from idealized Arrhenius Law behavior. When we model multi-step reactions of idealized chemical networks, we are unable to generate comparable deviations from linearity. In contrast, we find the two enzymes GAPDH and ß-galactosidase show non-linearity in the Arrhenius plot similar to our observations of embryonic development. Thus, we find that complex embryonic development can be well approximated by the simple Arrhenius equation regardless of non-uniform developmental scaling and propose that the observed departure from this law likely results more from non-idealized individual steps rather than from the complexity of the system.
Assuntos
TemperaturaRESUMO
Since the seminal 1961 paper of Monod and Jacob, mathematical models of biomolecular circuits have guided our understanding of cell regulation. Model-based exploration of the functional capabilities of any given circuit requires systematic mapping of multidimensional spaces of model parameters. Despite significant advances in computational dynamical systems approaches, this analysis remains a nontrivial task. Here, we use a nonlinear system of ordinary differential equations to model oocyte selection in Drosophila, a robust symmetry-breaking event that relies on autoregulatory localization of oocyte-specification factors. By applying an algorithmic approach that implements symbolic computation and topological methods, we enumerate all phase portraits of stable steady states in the limit when nonlinear regulatory interactions become discrete switches. Leveraging this initial exact partitioning and further using numerical exploration, we locate parameter regions that are dense in purely asymmetric steady states when the nonlinearities are not infinitely sharp, enabling systematic identification of parameter regions that correspond to robust oocyte selection. This framework can be generalized to map the full parameter spaces in a broad class of models involving biological switches.