Michael Veeman

chongmague reveals an essential role for laminin-mediated boundary formation in chordate convergence and extension movements

Although cell intercalation driven by non-canonical Wnt/planar cell polarity (PCP) pathway-dependent mediolateral cell polarity is important for notochord morphogenesis, it is likely that multiple mechanisms shape the notochord as it converges and extends. Here we show that the recessive short-tailed Ciona savignyi mutation chongmague (chm) has a novel defect in the formation of a morphological boundary around the developing notochord. chm notochord cells initiate intercalation normally, but then fail to maintain their polarized cell morphology and migrate inappropriately to become dispersed in the larval tail. This is unlike aimless (aim), a mutation in the PCP pathway component Prickle, which has a severe defect in early mediolateral intercalation but forms a robust notochord boundary. Positional cloning identifies chm as a mutation in the C. savignyi ortholog of the vertebrate alpha 3/4/5 family of laminins. Cs-lamα3/4/5 is highly expressed in the developing notochord, and Cs-lamα3/4/5 protein is specifically localized to the outer border of the notochord. Notochord convergence and extension, reduced but not absent in both chm and aim, are essentially abolished in the aim/aim; chm/chm double mutant, indicating that laminin-mediated boundary formation and PCP-dependent mediolateral intercalation are each able to drive a remarkable degree of tail morphogenesis in the absence of the other. These mechanisms therefore initially act in parallel, but we also find that PCP signaling has an important later role in maintaining the perinotochordal/intranotochordal polarity of Cs-lamα3/4/5 localization.

The ascidian mouth opening is derived from the anterior neuropore: reassessing the mouth/neural tube relationship in chordate evolution.

The relative positions of the brain and mouth are of central importance for models of chordate evolution. The dorsal hollow neural tube and the mouth have often been thought of as developmentally distinct structures that may have followed independent evolutionary paths. In most chordates however, including vertebrates and ascidians, the mouth primordia have been shown to fate to the anterior neural boundary. In ascidians such as Ciona there is a particularly intimate relationship between brain and mouth development, with a thin canal connecting the neural tube lumen to the mouth primordium at larval stages. This so-called neurohypophyseal canal was previously thought to be a secondary connection that formed relatively late, after the independent formation of the mouth primordium and the neural tube. Here we show that the Ciona neurohypophyseal canal is present from the end of neurulation and represents the anteriormost neural tube, and that the future mouth opening is actually derived from the anterior neuropore. The mouth thus forms at the anterior midline transition between neural tube and surface ectoderm. In the vertebrate Xenopus, we find that although the mouth primordium is not topologically continuous with the neural tube lumen, it nonetheless forms at this same transition point. This close association between the mouth primordium and the anterior neural tube in both ascidians and amphibians suggests that the evolution of these two structures may be more closely linked than previously appreciated.

doublesex/mab3 related-1 (dmrt1) is essential for development of anterior neural plate derivatives in Ciona.

Ascidian larvae have a hollow, dorsal central nervous system that shares many morphological features with vertebrate nervous systems yet is composed of very few cells. We show here that a null mutation in the gene dmrt1 in the ascidian Ciona savignyi results in profound abnormalities in the development of the sensory vesicle (brain), as well as other anterior ectodermal derivatives, including the palps and oral siphon primordium (OSP). Although the phenotype of the mutant embryos is variable, the majority have a complete loss of the most anterior structures (palps and OSP) and extensive disruption of sensory structures, such as the light-sensitive ocellus, in the sensory vesicle. dmrt1 is expressed early in the blastula embryo in a small group of presumptive ectodermal cells as they become restricted to anterior neural, OSP and palp fates. Despite the early and restricted expression of dmrt1, we were unable, using several independent criteria, to observe a defect in the mutant embryos until the early tailbud stage. We speculate that the variability and late onset in the phenotype may be due to partially overlapping activities of other gene products.

Whole-organ cell shape analysis reveals the developmental basis of ascidian notochord taper.

Here we use in toto imaging together with computational segmentation and analysis methods to quantify the shape of every cell at multiple stages in the development of a simple organ: the notochord of the ascidian Ciona savignyi. We find that cell shape in the intercalated notochord depends strongly on anterior-posterior (AP) position, with cells in the middle of the notochord consistently wider than cells at the anterior or posterior. This morphological feature of having a tapered notochord is present in many chordates. We find that ascidian notochord taper involves three main mechanisms: Planar Cell Polarity (PCP) pathway-independent sibling cell volume asymmetries that precede notochord cell intercalation; the developmental timing of intercalation, which proceeds from the anterior and posterior towards the middle; and the differential rates of notochord cell narrowing after intercalation. A quantitative model shows how the morphology of an entire developing organ can be controlled by this small set of cellular mechanisms.

Reciprocal and dynamic polarization of planar cell polarity core components and myosin

The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization. DOI: http://dx.doi.org/10.7554/eLife.05361.001

Exploiting the Extraordinary Genetic Polymorphism of Ciona for Developmental Genetics with Whole Genome Sequencing

Studies in tunicates such as Ciona have revealed new insights into the evolutionary origins of chordate development. Ciona populations are characterized by high levels of natural genetic variation, between 1 and 5%. This variation has provided abundant material for forward genetic studies. In the current study, we make use of deep sequencing and homozygosity mapping to map spontaneous mutations in outbred populations. With this method we have mapped two spontaneous developmental mutants. In Ciona intestinalis we mapped a short-tail mutation with strong phenotypic similarity to a previously identified mutant in the related species Ciona savignyi. Our bioinformatic approach mapped the mutation to a narrow interval containing a single mutated gene, α-laminin3,4,5, which is the gene previously implicated in C. savignyi. In addition, we mapped a novel genetic mutation disrupting neural tube closure in C. savignyi to a T-type Ca2+ channel gene. The high efficiency and unprecedented mapping resolution of our study is a powerful advantage for developmental genetics in Ciona, and may find application in other outbred species.

An automatic feature based model for cell segmentation from confocal microscopy volumes

We present a model for the automated segmentation of cells from confocal microscopy volumes of biological samples. The segmentation task for these images is exceptionally challenging due to weak boundaries and varying intensity during the imaging process. To tackle this, a two step pruning process based on the Fast Marching Method is first applied to obtain an over-segmented image. This is followed by a merging step based on an effective feature representation. The algorithm is applied on two different datasets: one from the ascidian Ciona and the other from the plant Arabidopsis. The presented 3D segmentation algorithm shows promising results on these datasets.

Segmentation of ascidian notochord cells in DIC timelapse images.

We have developed a method to automatically segment notochord cell boundaries from differential interference contrast (DIC) timelapse images of the elongating ascidian tail. The method is based on a specialized parametric active contour, the network snake, which can be initialized as a network of arbitrary but fixed topology and provides an effective framework for simultaneously segmenting multiple touching cells. Several modifications to the original network snake were necessary for high‐quality segmentation, including linear Gaussian derivative filtering to reconstruct edge maps from DIC images and a new energy function to improve the segmentation of critical cell‐cell vertices. We find that post‐intercalation ascidian notochord cells exhibit two distinct cell behaviors: lateral cell edges expand along the AP axis while showing a rapid pulsatile behavior, whereas anterior and posterior cell edges contract smoothly. Microsc. Res. Tech., 2011.

A linear program formulation for the segmentation of Ciona membrane volumes.

We address the problem of cell segmentation in confocal microscopy membrane volumes of the ascidian Ciona used in the study of morphogenesis. The primary challenges are non-uniform and patchy membrane staining and faint spurious boundaries from other organelles (e.g. nuclei). Traditional segmentation methods incorrectly attach to faint boundaries producing spurious edges. To address this problem, we propose a linear optimization framework for the joint correction of multiple over-segmentations obtained from different methods. The main idea motivating this approach is that multiple over-segmentations, resulting from a pool of methods with various parameters, are likely to agree on the correct segment boundaries, while spurious boundaries are methodor parameter-dependent. The challenge is to make an optimized decision on selecting the correct boundaries while discarding the spurious ones. The proposed unsupervised method achieves better performance than state of the art methods for cell segmentation from membrane images.

A curvicylindrical coordinate system for the visualization and segmentation of the ascidian tail

State of the art biological imaging methods, such as confocal microscopy, create 3D volumes by sampling on a cartesian grid. This cartesian coordinate system is often not convenient for visualization and analysis of multi layered organs or tissues. The ascidian embryonic tail, for example, is organized along anterioposterior (AP), dorsoventral (DV) and left-right (LR) axes that are locally orthogonal but curved in the XYZ microscope space. Here, we propose a “curvicylindrical” coordinate system for analysis of such biological structures. By extracting representative paths that traverse different tissue layers, the embryo can be visualized in a small number of 2D images (3 images in the case of the ascidian tail). As we demonstrate, this reduction of the dimensionality from 3D to 2D facilitates the initialization process for high quality segmentation of different cell types, and identification of tissue boundaries.