François B. Robin

Sequential Activation of Apical and Basolateral Contractility Drives Ascidian Endoderm Invagination

BACKGROUNDnEpithelial 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.nnnRESULTSnWe 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 because of Rho/Rho-kinase-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 basolateral enrichment of 1P-myosin drives apico-basal shortening, whereas 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.nnnCONCLUSIONSnOur 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.

A Multicassette Gateway Vector Set for High Throughput and Comparative Analyses in Ciona and Vertebrate Embryos

Background The 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 Findings To 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. Conclusion This 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.

The ANISEED database: Digital representation, formalization, and elucidation of a chordate developmental program

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.

Sequential Contraction and Exchange of Apical Junctions Drives Zippering and Neural Tube Closure in a Simple Chordate

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.

Single-molecule analysis of cell surface dynamics in Caenorhabditis elegans embryos

We describe a general, versatile and minimally invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in Caenorhabditis elegans embryos. We exploited 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). We introduce a method called smPReSS, single-molecule photobleaching relaxation to steady state, that infers exchange rates from quantitative analysis of single-molecule photobleaching kinetics without using SPT. Combining SPT and smPReSS allowed for spatially and temporally resolved measurements of protein mobility and exchange kinetics. We used these methods to (i) resolve distinct mobility states and spatial variation in exchange rates of the polarity protein PAR-6 and (ii) measure spatiotemporal modulation of actin filament assembly and disassembly. These methods offer a promising avenue to investigate dynamic mechanisms that pattern the embryonic cell surface.

Time-lapse imaging of live Phallusia embryos for creating 3D digital replicas.

During embryonic development, cell behaviors that are tightly coordinated both spatially and temporally integrate at the tissue level and drive embryonic morphogenesis. Over the past 20 years, advances in imaging techniques, in particular, the development of confocal imaging, have opened a new world in biology, not only giving us access to a wealth of information, but also creating new challenges. It is sometimes difficult to make the best use of the recordings of the complex, inherently three-dimensional (3D) processes we now can observe. In particular, these data are often not directly suitable for even simple but conceptually fundamental quantifications. This article provides a method to fluorescently label and image structures of interest that will subsequently be reconstructed, such as cell membranes or nuclei. The protocol describes live imaging of Phallusia mammillata embryos, which are robust, colorless, and optically transparent with negligible autofluorescence. Their diameter ranges from 100 µm to 120 µm, which allows time-lapse microscopy of whole embryos using two-photon microscopy with a high-resolution objective. Although two-photon imaging is described in detail, any imaging technology that results in a z-stack may be used. The resulting image stacks can subsequently be digitalized and segmented to produce 3D embryo replicas that can be interfaced to a model organism database and used to quantify cell shapes.

Excitable RhoA dynamics drive pulsed contractions in the early C. elegans embryo

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.

Creating 3D digital replicas of ascidian embryos from stacks of confocal images.

During embryonic development, cell behaviors that are tightly coordinated both spatially and temporally integrate at the tissue level and drive embryonic morphogenesis. Over the past 20 years, advances in imaging techniques, in particular, the development of confocal imaging, have opened a new world in biology, not only giving us access to a wealth of information, but also creating new challenges. It is sometimes difficult to make the best use of the recordings of the complex, inherently three-dimensional (3D) processes we now can observe. In particular, these data are often not directly suitable for even simple but conceptually fundamental quantifications. This article describes a process whereby image stacks gathered from live or fixed ascidian embryos are digitalized and segmented to produce 3D embryo replicas. These replicas can then be interfaced via a 3D Virtual Embryo module to a model organism database (Aniseed) that allows one to relate the geometrical properties of cells and cell contacts to additional parameters such as cell lineage, cell fates, or the underlying genetic program. Such an integrated system can serve several general purposes. First, it makes it possible to quantify and better understand the dynamics of cell behaviors during embryonic development, including, for instance, the automatic detection of asymmetric cell divisions or the evolution of cell contacts. Second, the 3D Virtual Embryo software proposes a panel of mathematical shape descriptors to precisely quantify cellular geometries and generate a 3D identity card for each embryonic cell. Such reconstructions open the door to a detailed 3D simulation of morphogenesis.

Imaging of fixed ciona embryos for creating 3D digital replicas.

During embryonic development, cell behaviors that are tightly coordinated both spatially and temporally integrate at the tissue level and drive embryonic morphogenesis. Over the past 20 years, advances in imaging techniques, in particular, the development of confocal imaging, have opened a new world in biology, not only giving us access to a wealth of information, but also creating new challenges. It is sometimes difficult to make the best use of the recordings of the complex, inherently three-dimensional (3D) processes we now can observe. In particular, these data are often not directly suitable for even simple but conceptually fundamental quantifications. This article presents a method for imaging embryonic development with cellular resolution in fixed ascidian embryos. A large fraction of the ascidian community primarily studies the development of the cosmopolitan ascidian Ciona intestinalis. Because the embryos of this species are insufficiently transparent and show significant autofluorescence, live imaging is difficult. Thus, whole embryos are fixed and optically cleared. They are then stained and imaged on a regular or two-photon confocal microscope. The resulting image stacks can subsequently be digitalized and segmented to produce 3D embryo replicas that can be interfaced to a model organism database and used to quantify cell shapes.