A model-based approach to designing developmental toxicology experiments using sea urchin embryos

The key aim of this paper is to suggest a more quantitative approach to designing a dose–response experiment, and more specifically, a concentration–response experiment. The work proposes a departure from the traditional experimental design to determine a dose–response relationship in a developmental toxicology study. It is proposed that a model-based approach to determine a dose–response relationship can provide the most accurate statistical inference for the underlying parameters of interest, which may be estimating one or more model parameters or pre-specified functions of the model parameters, such as lethal dose, at maximal efficiency. When the design criterion or criteria can be determined at the onset, there are demonstrated efficiency gains using a more carefully selected model-based optimal design as opposed to an ad-hoc empirical design. As an illustration, a model-based approach was theoretically used to construct efficient designs for inference in a developmental toxicity study of sea urchin embryos exposed to trimethoprim. This study compares and contrasts the results obtained using model-based optimal designs versus an ad-hoc empirical design.

Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network

The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament-like network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide the visualization of an apparent septin filament network with the CR structure of an animal cell.

V-type H+ ATPase Activity is Required for Embryonic Dorsal-Ventral Symmetry Breaking

The mechanism for embryonic dorsal-ventral (DV) symmetry breaking is idiosyncratic to the species, then converges on polarized expression of BMP signaling ligands. Here, we show that V-ATPase (VHA) activity is an early requirement for DV symmetry breaking in sea urchin embryos. In these basal deuterostomes, DV specification is mediated by ventral Nodal expression that leads to the establishment of a BMP signaling gradient. Nodal expression occurs downstream from p38 MAPK, which is transiently asymmetrically active. We show that VHA activity is required for DV symmetry breaking upstream from both p38 MAPK and Nodal. We rescue VHA-mediated ventralization by enforcing Nodal signaling asymmetry. We identify a VHA-dependent DV voltage gradient and also find that VHA activity is required for hypoxia inducible factor (HIF) activation. However, neither hyperpolarization nor HIF activation account for the dorsalizing effects of VHA, implicating a third unknown pathway that connects VHA activity to p38 MAPK symmetry breaking.

H+/K+ Ion Pump Enhances Cytoskeletal Polarity to Drive Gastrulation in Sea Urchin Embryo

Gastrulation is a universal process in the morphogenesis of many animal embryos. In sea urchin embryos, it involves the invagination of a single-layered vegetal plate into the blastocoel. Although morphological and molecular events in gastrulation have been well studied, the mechanical driving forces and the regulatory mechanism underlying gastrulation is not fully understood. In this study, structural features and cytoskeletal distributions were studied in sea urchin embryos using an “exogastrulation” model induced by inhibiting the H+/K+ ion pump with omeprazole. The vegetal poles of the exogastrulating embryos showed reduced roundness indices, intracellular pH polarization, and intracellular F-actin polarization at the pre-early gastrulation stage compared with normal embryos. Gastrulation stopped when F-actin polymerization or degradation was inhibited via RhoA or YAP1 knockout, although pH distributions were independent of such a knockout. A mathematical model of sea urchin embryos at the early gastrulation reproduced the shapes of both normal and exogastrulating embryos using cell-dependent cytoskeletal features based on F-actin and pH distributions. Thus, gastrulation required appropriate cell position-dependent intracellular F-actin distributions regulated by the H+/K+ ion pump through pH control.

H+/K+ ion pump enhances cytoskeletal polarity to drive gastrulation in sea urchin embryo

Gastrulation is a universal process in the morphogenesis of many animal embryos. In sea urchin embryos, it involves the invagination of single-layered vegetal plate into blastocoel. Although morphological and molecular events have been well studied for gastrulation, the mechanical driving forces and their regulatory mechanism underlying the gastrulation is not fully understood. In this study, structural features and cytoskeletal distributions were studied in sea urchin embryo using an "exogastrulation" model induced by inhibiting the H+/K+ ion pump with omeprazole. The vegetal pole sides of the exogastrulating embryos had reduced roundness indices, intracellular pH polarization, and intracellular F-actin polarization at the pre-early gastrulation compared with the normal embryo. Gastrulation stopped when F-actin polymerization or degradation was inhibited by RhoA or YAP1 knockout, although pH distributions were independent of such a knockout. A mathematical model of sea urchin embryos at the early gastrulation reproduced the shapes of both normal and exogastrulating embryos using cell-dependent cytoskeletal features based on F-actin and pH distributions. Thus, gastrulation required appropriate cell position-dependent intracellular F-actin distributions regulated by the H+/K+ ion pump through pH control.