SUMMARY Background Epithelial invagination is a fundamental morphogenetic behavior that transforms a flat cell sheet into a pit or groove. Previous studies of invagination have focused on the role of actomyosin-dependent apical contraction; other mechanisms remain largely unexplored. Results We combined experimental and computational approaches to identify a two-step mechanism for endoderm invagination during ascidian gastrulation. During Step 1, which immediately precedes invagination, endoderm cells constrict their apices due to Rho/Rhokinase-dependent apical enrichment of 1P–myosin. Our data suggest that endoderm invagination itself occurs during Step 2, without further apical shrinkage, via a novel mechanism we call collared rounding: Rho/Rho-kinase-independent lateral enrichment of 1P–myosin drives apico-basal shortening, while Rho/Rho-kinase-dependent enrichment of 1P and 2P myosin in circumapical collars is required to prevent apical expansion and for deep invagination. Simulations show that boundary-specific tension values consistent with these distributions of active myosin can explain the cell shape changes observed during invagination both in normal embryos and in embryos treated with pharmacological inhibitors of either Rho-kinase or Myosin II ATPase. Indeed, we find that the balance of strong circumapical and basolateral tension is the only mechanism based on differential cortical tension that can explain ascidian endoderm invagination. Finally, simulations suggest that mesectoderm cells resist endoderm shape changes during both steps and we confirm this prediction experimentally. Conclusions Our findings suggest that early ascidian gastrulation is driven by the coordinated apposition of circumapical and lateral endoderm contraction, working against a resisting mesectoderm. We propose that similar mechanisms may operate during other invaginations.
Pulsed actomyosin contractility underlies diverse modes of tissue morphogenesis, but the underlying mechanisms remain poorly understood. Here, we combined quantitative imaging with genetic perturbations to identify a core mechanism for pulsed contractility in early Caenorhabditis elegans embryos. We show that pulsed accumulation of actomyosin is governed by local control of assembly and disassembly downstream of RhoA. Pulsed activation and inactivation of RhoA precede, respectively, the accumulation and disappearance of actomyosin and persist in the absence of Myosin II. We find that fast (likely indirect) autoactivation of RhoA drives pulse initiation, while delayed, F-actin–dependent accumulation of the RhoA GTPase-activating proteins RGA-3/4 provides negative feedback to terminate each pulse. A mathematical model, constrained by our data, suggests that this combination of feedbacks is tuned to generate locally excitable RhoA dynamics. We propose that excitable RhoA dynamics are a common driver for pulsed contractility that can be tuned or coupled differently to actomyosin dynamics to produce a diversity of morphogenetic outcomes.
We describe a general, versatile and non-invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in C. elegans embryos. We exploit tunable expression via RNAi and a dynamically exchanging monomer pool to achieve fast continuous single-molecule imaging at optimal densities with signal-to-noise ratios adequate for robust single particle tracking (SPT) analysis. We also introduce and validate a new method called smPReSS that infers exchange rates from quantitative analysis of single molecule photobleaching kinetics, without using SPT. Combining SPT and smPReSS allows spatially and temporally resolved measurements of protein mobility and exchange kinetics. We use these methods (a) to resolve distinct mobility states and spatial variation in exchange rates of the polarity protein Par-6 and (b) to measure spatiotemporal modulation of actin filament assembly and disassembly. The introduction of these methods in a powerful model system offers a promising new avenue to investigate dynamic mechanisms that pattern the embryonic cell surface.
Unidirectional zippering is a key step in neural tube closure that remains poorly understood. Here, we combine experimental and computational approaches to identify the mechanism for zippering in a basal chordate, Ciona intestinalis. We show that myosin II is activated sequentially from posterior to anterior along the neural/epidermal (Ne/Epi) boundary just ahead of the advancing zipper. This promotes rapid shortening of Ne/Epi junctions, driving the zipper forward and drawing the neural folds together. Cell contact rearrangements (Ne/Epi + Ne/Epi → Ne/Ne + Epi/Epi) just behind the zipper lower tissue resistance to zipper progression by allowing transiently stretched cells to detach and relax toward isodiametric shapes. Computer simulations show that measured differences in junction tension, timing of primary contractions, and delay before cell detachment are sufficient to explain the speed and direction of zipper progression and highlight key advantages of a sequential contraction mechanism for robust efficient zippering.
BackgroundThe past few years have seen a vast increase in the amount of genomic data available for a growing number of taxa, including sets of full length cDNA clones and cis-regulatory sequences. Large scale cross-species comparisons of protein function and cis-regulatory sequences may help to understand the emergence of specific traits during evolution.Principal FindingsTo facilitate such comparisons, we developed a Gateway compatible vector set, which can be used to systematically dissect cis-regulatory sequences, and overexpress wild type or tagged proteins in a variety of chordate systems. It was developed and first characterised in the embryos of the ascidian Ciona intestinalis, in which large scale analyses are easier to perform than in vertebrates, owing to the very efficient embryo electroporation protocol available in this organism. Its use was then extended to fish embryos and cultured mammalian cells.ConclusionThis versatile vector set opens the way to the mid- to large-scale comparative analyses of protein function and cis-regulatory sequences across chordate evolution. A complete user manual is provided as supplemental material.
Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions.
Pulsed actomyosin contractility underlies diverse modes of tissue morphogenesis, but the underlying mechanisms remain poorly understood. Here, we combine quantitative imaging with genetic perturbations to identify a core mechanism for pulsed contractility in early C. elegans embryos. We show that pulsed accumulation of actomyosin is governed by local control of assembly and disassembly downstream of RhoA. Pulsed activation and inactivation of RhoA precede, respectively, accumulation and disappearance of actomyosin, and persist in the nearly complete absence of Myosin II.We find that fast positive feedback on RhoA activation drives pulse initiation, while Factin dependent accumulation of the RhoA GTPase activating proteins (GAPs) RGA-3/4 provides delayed negative feedback to terminate each pulse. An experimentally constrained mathematical model confirms that in principle these feedbacks are sufficient to generate locally excitable RhoA dynamics. We propose that excitable RhoA dynamics are a common driver for pulsed contractility that can be differently tuned or coupled to actomyosin dynamics to produce a diversity of morphogenetic outcomes.
6This 8-week, randomized, double-blind, controlled study compared efficacy and tolerability of telmisartan ⁄ amlodipine (T ⁄ A) single-pill combination (SPC) vs the respective monotherapies in 858 patients with severe hypertension (systolic ⁄ diastolic blood pressure [SBP ⁄ DBP] !180 ⁄ 95 mm Hg). At 8 weeks, T ⁄ A provided significantly greater reductions from baseline in seated trough cuff SBP ⁄ DBP ()47.5 mm Hg ⁄ )18.7 mm Hg) vs T (P<.0001) or A (P=.0002) monotherapy; superior reductions were also evident at 1, 2, 4, and 6 weeks. Blood pressure (BP) goal and response rates were consistently higher with T ⁄ A vs T or A. T ⁄ A was well tolerated, with less frequent treatmentrelated adverse events vs A (12.6% vs 16.4%) and a numerically lower incidence of peripheral edema and treatment discontinuation. In conclusion, treatment of patients with substantially elevated BP with T ⁄ A SPCs resulted in high and significantly greater BP reductions and higher BP goal and response rates than the respective monotherapies. T ⁄ A SPCs were well tolerated. J Clin Hypertens (Greenwich). 2012;14:206-215. Ó2012 Wiley Periodicals, Inc.Based on evidence from a number of large antihypertensive trials, 1-9 most guidelines acknowledge that combination therapy is needed to reduce blood pressure (BP) successfully to goal in the majority of patients; only a minority of patients achieve their BP goal with a single agent.10-14 Also, the Avoiding Cardiovascular Events Through Combination Therapy in Patients Living With Systolic Hypertension (ACCOMPLISH) study showed a significant reduction of cardiovascular (CV) events and death in hypertensive patients at high CV risk treated with a combination of an angiotensin-converting enzyme (ACE) inhibitor and a calcium channel blocker (CCB).15 Nevertheless, despite rigorous and comprehensive guidelines, and a trend towards an increase in the use of combination therapy in treatment practice, 16 several studies have demonstrated the persistence of poor BP goal rates in treated patients. [17][18][19] The impact of poor BP control is compounded by the often high prevalence of other CV risk factors in hypertensive patients (eg, hypercholesterolemia, obesity, type 2 diabetes mellitus [T2DM], and smoking).13 Therefore, an urgent need still remains to improve the management of hypertension. One logical approach would be to use 2 drugs from different classes and complementary mechanisms of action in combination. Such combinations may result in additional BP decreases and improved goal rates, compared with either agent used alone. 20-23Furthermore, single-pill combinations (SPCs) are known to increase treatment adherence and reduce health care costs. [24][25][26][27] A combination of a CCB and an angiotensin II receptor blocker (ARB) is a rational approach for managing hypertension and there is increasing evidence that this combination is effective. 11,13,28,29 The aim of the current study was to compare the efficacy and tolerability of the SPC of telmisartan 80 mg ⁄ amlodipine 10 mg (T80 ⁄ A10) with that of...
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