During embryonic development, temporal and spatial cues are coordinated to generate a segmented body axis. In sequentially segmenting animals, the rhythm of segmentation is reported to be controlled ...by the time scale of genetic oscillations that periodically trigger new segment formation. However, we present real-time measurements of genetic oscillations in zebrafish embryos showing that their time scale is not sufficient to explain the temporal period of segmentation. A second time scale, the rate of tissue shortening, contributes to the period of segmentation through a Doppler effect. This contribution is modulated by a gradual change in the oscillation profile across the tissue. We conclude that the rhythm of segmentation is an emergent property controlled by the time scale of genetic oscillations, the change of oscillation profile, and tissue shortening.
Synthetic gene oscillators have the potential to control timed functions and periodic gene expression in engineered cells. Such oscillators have been refined in bacteria in vitro, however, these ...systems have lacked the robustness and precision necessary for applications in complex in vivo environments, such as the mammalian gut. Here, we demonstrate the implementation of a synthetic oscillator capable of keeping robust time in the mouse gut over periods of days. The oscillations provide a marker of bacterial growth at a single-cell level enabling quantification of bacterial dynamics in response to inflammation and underlying variations in the gut microbiota. Our work directly detects increased bacterial growth heterogeneity during disease and differences between spatial niches in the gut, demonstrating the deployment of a precise engineered genetic oscillator in real-life settings.
Thoracic aortic aneurysm (TAA) is a potentially lethal condition, which can affect individuals of all ages. TAA may be complicated by the sudden onset of life-threatening dissection or rupture. The ...underlying mechanisms leading to TAA formation, particularly in the nonsyndromal idiopathic group of patients, are not well understood. Thus, identification of new genes and targets that are involved in TAA pathogenesis are required to help prevent and reverse the disease phenotype.
Here we explore the role of ARHGAP18, a novel Rho GAP expressed by smooth muscle cells (SMCs), in the pathogenesis of TAA.
Using human and mouse aortic samples, we report that ARHGAP18 levels were significantly reduced in the SMC layer of aortic aneurysms.
global knockout (
) mice exhibited a highly synthetic, proteolytic, and proinflammatory smooth muscle phenotype under basal conditions and when challenged with angiotensin II, developed TAA with increased frequency and severity compared with littermate controls. Chromatin immunoprecipitation studies revealed this phenotype is partly associated with strong enrichment of H3K4me3 and depletion of H3K27me3 at the
and
promoters in
-deficient SMC. We further show that TAA formation in the
mice is associated with loss of Akt activation. The abnormal SMC phenotype observed in the
mice can be partially rescued by pharmacological treatment with the mTORC1 inhibitor rapamycin, which reduces the synthetic and proinflammatory phenotype of
-deficient SMC.
We have identified
as a novel protective gene against TAA formation and define an additional target for the future development of treatments to limit TAA pathogenesis.
Growing knowledge of the key molecular components involved in biological processes such as endocytosis, exocytosis, and motility has enabled direct testing of proposed mechanistic models by ...reconstitution. However, current techniques for building increasingly complex cellular structures and functions from purified components are limited in their ability to create conditions that emulate the physical and biochemical constraints of real cells. Here we present an integrated method for forming giant unilamellar vesicles with simultaneous control over (i) lipid composition and asymmetry, (ii) oriented membrane protein incorporation, and (iii) internal contents. As an application of this method, we constructed a synthetic system in which membrane proteins were delivered to the outside of giant vesicles, mimicking aspects of exocytosis. Using confocal fluorescence microscopy, we visualized small encapsulated vesicles docking and mixing membrane components with the giant vesicle membrane, resulting in exposure of previously encapsulated membrane proteins to the external environment. This method for creating giant vesicles can be used to test models of biological processes that depend on confined volume and complex membrane composition, and it may be useful in constructing functional systems for therapeutic and biomaterials applications.
Abstract Bicuspid aortic valve (BAV) is the commonest congenital heart disease and a highly heritable trait; however, only the NOTCH1 gene has been linked to limited cases of BAV in humans. Recently, ...the transcription factor GATA5 has been shown to have an essential role in aortic valve development, and targeted deletion of Gata5 in mice is associated with partially penetrant BAV formation. Here, we investigated the relationship between GATA5 gene variants and BAV with its associated aortopathy. One hundred unrelated individuals with confirmed BAV were prospectively recruited. Following collection of clinical information and DNA extraction, the coding regions and splice signal sequences of the GATA5 gene were screened for sequence variations. The clinical characteristics of the cohort included a male predominance (77%), mean age of diagnosis 29 ± 22 years, associated aortopathy in 59% and positive family history for BAV in 13%. Genetic analysis identified the presence of 4 rare non-synonymous variations within the GATA5 transcriptional activation domains, namely Gln3Arg, Ser19Trp, Tyr142His and Gly166Ser, occurring in one patient each. Gln3Arg and Tyr142His substitutions affect highly conserved and functionally relevant residues, and are likely to impact on the transcriptional activation of GATA5 target regions. A novel Ser19Trp variation was identified at a highly conserved amino acid residue in one patient, while the Gly166Ser variant was found in a familial case of BAV and associated aortopathy. Rare non-synonymous variations in the functionally important GATA5 transcriptional activation domains may be important in the pathogenesis of BAV disease in humans.
Little is known about how the sizes of animal tissues are controlled. A prominent example is somite size, which varies widely both within an individual and across species. Despite intense study of ...the segmentation clock governing the timing of somite generation, how it relates to somite size is poorly understood. Here, we examine somite scaling and find that somite size at specification scales with the length of the presomitic mesoderm (PSM) despite considerable variation in PSM length across developmental stages and in surgically size-reduced embryos. Measurement of clock period, axis elongation speed and clock gene expression patterns demonstrate that existing models fail to explain scaling. We posit a 'clock and scaled gradient' model, in which somite boundaries are set by a dynamically scaling signaling gradient across the PSM. Our model not only explains existing data, but also makes a unique prediction that we confirm experimentally - the formation of periodic 'echoes' in somite size following perturbation of the size of one somite. Our findings demonstrate that gradient scaling plays a central role in both progression and size control of somitogenesis.
Dynamic interplay between the plasma membrane and underlying cytoskeleton is essential for cellular shape change. Spatial organization of actin filaments, the growth of which generates membrane ...deformations during motility, phagocytosis, endocytosis and cytokinesis, is mediated by specific protein-protein interactions that branch, crosslink and bundle filaments into networks that interact with the membrane. Although membrane curvature has been found to influence binding of proteins with curvature-sensitive domains, the direct effect of membrane elasticity on cytoskeletal network organization is not clear. Here, we show through in vitro reconstitution and elastic modelling that a lipid bilayer can drive the emergence of bundled actin filament protrusions from branched actin filament networks, thus playing a role normally attributed to actin-binding proteins. Formation of these filopodium-like protrusions with only a minimal set of purified proteins points to an active participation of the membrane in organizing actin filaments at the plasma membrane. In this way, elastic interactions between the membrane and cytoskeleton can cooperate with accessory proteins to drive cellular shape change.
Compartmentalization of biomolecules within lipid membranes is a fundamental requirement of living systems and an essential feature of many pharmaceutical therapies. However, applications of ...membrane-enclosed solutions of proteins, DNA, and other biologically active compounds have been limited by the difficulty of forming unilamellar vesicles with controlled contents in a repeatable manner. Here, we demonstrate a method for simultaneously creating and loading giant unilamellar vesicles (GUVs) using a pulsed microfluidic jet. Akin to blowing a bubble, the microfluidic jet deforms a planar lipid bilayer into a vesicle that is filled with solution from the jet and separates from the planar bilayer. In contrast with existing techniques, our method rapidly generates multiple monodisperse, unilamellar vesicles containing solutions of unrestricted composition and molecular weight. Using the microfluidic jetting technique, we demonstrate repeatable encapsulation of 500-nm particles into GUVs and show that functional pore proteins can be incorporated into the vesicle membrane to mediate transport. The ability of microfluidic jetting to controllably encapsulate solutions inside of GUVs creates new opportunities for the study and use of compartmentalized biomolecular systems in science, industry, and medicine.
Metamerism is a widespread feature of multicellular body plans; however, our understanding of the underlying mechanisms that generate these patterns is currently based on only a few model organisms. ...In particular, vertebrate embryos use a segmentation clock to rhythmically and sequentially add segments in concert with posterior elongation of their body. Recent evidence of a segmentation clock acting in arthropods indicates that this mechanism may be a widely used strategy for generating serial anatomy in animals. Whether this is due to homology or convergence is not yet known, but the recent discovery of an oscillatory process associated with the production of sequential root primordia in plants suggests that a segmentation clock is a fundamental patterning principle in growing tissues, independent of ancestry. In this review, we consider the principles of the segmentation clock that may be conserved across the animal and plant kingdoms, and discuss opportunities for cross-fertilization between these active fields of research.
Signaling events that regulate central nervous system (CNS) angiogenesis and blood-brain barrier (BBB) formation are only beginning to be elucidated. By evaluating the gene expression profile of ...mouse vasculature, we identified DR6/TNFRSF21 and TROY/TNFRSF19 as regulators of CNS-specific angiogenesis in both zebrafish and mice. Furthermore, these two death receptors interact both genetically and physically and are required for vascular endothelial growth factor (VEGF)-mediated JNK activation and subsequent human brain endothelial sprouting in vitro. Increasing beta-catenin levels in brain endothelium upregulate DR6 and TROY, indicating that these death receptors are downstream target genes of Wnt/beta-catenin signaling, which has been shown to be required for BBB development. These findings define a role for death receptors DR6 and TROY in CNS-specific vascular development.
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► Angiogenesis and blood-brain barrier formation require death receptors DR6 and TROY ► DR6 and TROY physically and genetically interact ► DR6 and TROY regulate VEGF-induced JNK signaling in endothelial cells ► Wnt/beta-catenin signaling upregulates DR6 and TROY in brain endothelium
The signaling events that regulate central nervous system (CNS) angiogenesis and blood-brain barrier formation are only beginning to be elucidated. Tam et al. identify the TNF receptor family proteins DR6 and TROY as regulators of CNS-specific vascular development, acting cell-autonomously to modulate vascular sprouting and subsequent barrier formation.
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