Erin Newman-Smith

The maternal-to-zygotic transition in embryonic patterning of Caenorhabditis elegans.

Maternal factors laid down in the oocyte regulate blastomere identities in the early Caenorhabditis elegans embryo by activating zygotic patterning genes and restricting their expression to the appropriate lineages. A number of early-acting zygotic genes that specify various cell fates have been identified recently and their temporal and spatial regulation by maternal factors has begun to be elucidated.

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.

Functional analysis of trophoblast giant cells in parthenogenetic mouse embryos.

Diploid mouse embryos containing only maternal DNA (parthenotes) fail, in part, because the inner cell mass does not induce the trophoblast to grow. In this study, we asked whether any of the defects in parthenotes may arise from alterations in trophoblast function. We examined the expression of genes important for normal trophoblast function and found several trophoblast genes that were expressed at normal levels in the primary trophoblast cells of parthenotes: E-cadherin, a cell adhesion molecule, was expressed normally in both the ICM and trophectoderm of parthenogenetic blastocysts and blastocyst outgrowths; the gene for Hxt, a basic helix-loop-helix factor that regulates trophoblast development, was expressed in both zygotic and parthenogenetic giant cells; placental lactogen-1, a hormone that is normally secreted by trophoblast giant cells, was expressed in most of both parthenogenetic and normal trophoblast cells; and the 92 kDa matrix metalloproteinase, gelatinase B, also known as MMP-9, was secreted at equivalent levels by both zygotic and parthenogenetic blastocyst outgrowths. However, once the outgrowths had developed, a subpopulation of trophoblast cells in parthenogenetic embryos had decreased DNA replication and significantly fewer nucleoli per nucleus than did zygotic embryos. Moreover, the parthenogenetic trophoblast cells growing out from blastocysts had a decreased viability in culture. These data suggest that, although parthenogenetic embryos are able to initiate primary trophoblast differentiation, the stability and continued differentiation of trophoblast giant cells may be abnormal. Our data support the hypothesis that the deficiency of secondary trophoblast giant cells may contribute to the decline of parthenogenetic embryos and suggest that the factors controlling this subset of trophoblast are distinct from those for primary trophoblast.

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

Key steps in the morphogenesis of a cranial placode in an invertebrate chordate, the tunicate Ciona savignyi

Tunicates and vertebrates share a common ancestor that possessed cranial neurogenic placodes, thickenings in embryonic head epidermis giving rise to sensory structures. Though orthology assignments between vertebrate and tunicate placodes are not entirely resolved, vertebrate otic placodes and tunicate atrial siphon primordia are thought to be homologous based on morphology and position, gene expression, and a common signaling requirement during induction. Here, we probe key points in the morphogenesis of the tunicate atrial siphon. We show that the siphon primordium arises within a non-dividing field of lateral-dorsal epidermis. The initial steps of atrial primordium invagination are similar to otic placode invagination, but a placode-derived vesicle is never observed as for the otic vesicle of vertebrates. Rather, confocal imaging reveals an atrial opening through juvenile stages and beyond. We inject a photoactivatable lineage tracer to show that the early atrial siphon of the metamorphic juvenile, including its aperture and lining, derives from cells of the atrial placode itself. Finally, we perturb the routing of the gut to the left atrium by laser ablation and pharmacology to show that this adaptation to a sessile lifestyle depends on left-right patterning mechanisms present in the free-swimming chordate ancestor.

A one-dimensional model of PCP signaling: Polarized cell behavior in the notochord of the ascidian Ciona

Despite its importance in development and physiology the planar cell polarity (PCP) pathway remains one of the most enigmatic signaling mechanisms. The notochord of the ascidian Ciona provides a unique model for investigating the PCP pathway. Interestingly, the notochord appears to be the only embryonic structure in Ciona activating the PCP pathway. Moreover, the Ciona notochord as a single-file array of forty polarized cells is a uniquely tractable system for the study of polarization dynamics and the transmission of the PCP pathway. Here, we test models for propagation of a polarizing signal, interrogating temporal, spatial and signaling requirements. A simple cell-cell relay cascading through the entire length of the notochord is not supported; instead a more complex mechanism is revealed, with interactions influencing polarity between neighboring cells, but not distant ones. Mechanisms coordinating notochord-wide polarity remain elusive, but appear to entrain general (i.e., global) polarity even while local interactions remain important. However, this global polarizer does not appear to act as a localized, spatially-restricted determinant. Coordination of polarity along the long axis of the notochord requires the PCP pathway, a role we demonstrate is temporally distinct from this pathways earlier role in convergent extension and intercalation. We also reveal polarity in the notochord to be dynamic: a cells polarity state can be changed and then restored, underscoring the Ciona notochords amenability for in vivo studies of PCP.

A transiently expressed connexin is essential for anterior neural plate development in Ciona intestinalis

A forward genetic screen in the ascidian Ciona intestinalis identified a mutant line (frimousse) with a profound disruption in neural plate development. In embryos with the frimousse mutation, the anteriormost neural plate cells, which are products of an FGF induction at the blastula and gastrula stages, initially express neural plate-specific genes but fail to maintain the induced state and ultimately default to epidermis. The genetic lesion in the frimousse mutant lies within a connexin gene (cx-11) that is transiently expressed in the developing neural plate in a temporal window corresponding to the period of a-lineage neural induction. Using a genetically encoded calcium indicator we observed multiple calcium transients throughout the developing neural plate in wild-type embryos, but not in mutant embryos. A series of treatments at the gastrula and neurula stages that block the calcium transients, including gap junction inhibition and calcium depletion, were also found to disrupt the development of the anterior neural plate in a similar way to the frimousse mutation. The requirement for cx-11 for anterior neural fate points to a crucial role for intercellular communication via gap junctions, probably through mediation of Ca2+ transients, in Ciona intestinalis neural induction.

CellECT: cell evolution capturing tool

BackgroundRobust methods for the segmentation and analysis of cells in 3D time sequences (3D+t) are critical for quantitative cell biology. While many automated methods for segmentation perform very well, few generalize reliably to diverse datasets. Such automated methods could significantly benefit from at least minimal user guidance. Identification and correction of segmentation errors in time-series data is of prime importance for proper validation of the subsequent analysis. The primary contribution of this work is a novel method for interactive segmentation and analysis of microscopy data, which learns from and guides user interactions to improve overall segmentation.ResultsWe introduce an interactive cell analysis application, called CellECT, for 3D+t microscopy datasets. The core segmentation tool is watershed-based and allows the user to add, remove or modify existing segments by means of manipulating guidance markers. A confidence metric learns from the user interaction and highlights regions of uncertainty in the segmentation for the user’s attention. User corrected segmentations are then propagated to neighboring time points. The analysis tool computes local and global statistics for various cell measurements over the time sequence. Detailed results on two large datasets containing membrane and nuclei data are presented: a 3D+t confocal microscopy dataset of the ascidian Phallusia mammillata consisting of 18 time points, and a 3D+t single plane illumination microscopy (SPIM) dataset consisting of 192 time points. Additionally, CellECT was used to segment a large population of jigsaw-puzzle shaped epidermal cells from Arabidopsis thaliana leaves. The cell coordinates obtained using CellECT are compared to those of manually segmented cells.ConclusionsCellECT provides tools for convenient segmentation and analysis of 3D+t membrane datasets by incorporating human interaction into automated algorithms. Users can modify segmentation results through the help of guidance markers, and an adaptive confidence metric highlights problematic regions. Segmentations can be propagated to multiple time points, and once a segmentation is available for a time sequence cells can be analyzed to observe trends. The segmentation and analysis tools presented here generalize well to membrane or cell wall volumetric time series datasets.

Photoreceptor specialization and the visuomotor repertoire of the primitive chordate Ciona

ABSTRACT The swimming tadpole larva of Ciona has one of the simplest central nervous systems (CNSs) known, with only 177 neurons. Despite its simplicity, the Ciona CNS has a common structure with the CNS of its close chordate relatives, the vertebrates. The recent completion of a larval Ciona CNS connectome creates enormous potential for detailed understanding of chordate CNS function, yet our understanding of Ciona larval behavior is incomplete. We show here that Ciona larvae have a surprisingly rich and dynamic set of visual responses, including a looming-object escape behavior characterized by erratic circular swims, as well as negative phototaxis characterized by sustained directional swims. Making use of mutant lines, we show that these two behaviors are mediated by distinct groups of photoreceptors. The Ciona connectome predicts that these two behavioral responses should act through distinct, but overlapping, visuomotor pathways, and that the escape behavior is likely to be integrated into a broader startle behavior. Highlighted Article: Ciona larvae looming-object escape behavior and negative phototaxis are mediated by different groups of photoreceptors and involve distinct, but overlapping, visuomotor pathways, and are characterized by circular and straight swim patterns, respectively.