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Defining pattern formation mechanisms during embryonic development is important for understanding the etiology of birth defects and to inform tissue engineering approaches. In this study, we used tricaine, a voltage-gated sodium channel (VGSC) inhibitor, to show that VGSC activity is required for normal skeletal patterning inLytechinus variegatussea urchin larvae. We demonstrate that tricaine-mediated patterning defects are rescued by an anesthetic-insensitive version of the VGSC LvScn5a. Expression of this channel is enriched in the ventrolateral ectoderm where it spatially overlaps with posterolaterally expressed Wnt5. We show that VGSC activity is required to spatially restrict Wnt5 expression to this ectodermal region that is adjacent and instructive to clusters of primary mesenchymal cells that initiate secretion of the larval skeleton as triradiates. Tricaine-mediated Wnt5 spatial expansion correlates with the formation of ectopic PMC clusters and triradiates. These defects are rescued by Wnt5 knock down, indicating that the spatial expansion Wnt5 is responsible for the patterning defects induced by VGSC inhibition. These results demonstrate a novel connection between bioelectrical status and the spatial control of patterning cue expression during embryonic pattern formation.Summary statementInhibition of voltage-gated sodium channels perturbs Wnt5-mediated patterning of the sea urchin larval skeleton

MotivationThe detection of distinct cellular identities is central to the analysis of single-cell RNA sequencing experiments. However, in perturbation experiments, current methods typically fail to correctly match cell states between conditions or erroneously remove population substructure. Here we present the novel, unsupervised algorithm ICAT that employs self-supervised feature weighting and control-guided clustering to accurately resolve cell states across heterogeneous conditions.ResultsUsing simulated and real datasets, we show ICAT is superior in identifying and resolving cell states compared to current integration workflows. While requiring no a priori knowledge of extant cell states or discriminatory marker genes, ICAT is robust to low signal strength, high perturbation severity, and disparate cell type proportions. We empirically validate ICAT in a developmental model and find that only ICAT identifies a perturbation-unique cellular response. Taken together, our results demonstrate that ICAT offers a significant improvement in defining cellular responses to perturbation in single-cell RNA sequencing data.Availability and implementationhttps://github.com/BradhamLab/icatSupplemental Methods, Tables and Figures are available online.

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.Graphical Abstract

Homeostasis of charged particles in biological systems is fundamental for life. Indeed, the efficient synthesis of ATP is predicated on an electrochemical gradient. Ion pumps and channels act as conduits that regulate membrane potential by controlling ion flux. This phenomenon is critical for the generation of action potentials in excitable cells such as neurons and muscle fibers, and for acidification of lysosomes in all cells. However, the production of action potentials or pH differentials is merely one facet of bioelectricity. Increasing evidence has shown that ion channels and pumps also play critical roles in other cellular processes: cell cycle regulation, wound healing, regeneration, and symmetry breaking during development. In recent years, the functional roles of ion channels have been explored in echinoderm development. The application of fluorescence-based ion- and voltage-sensitive dyes allows for live measurements of ion concentrations with both spatial and temporal resolution. In this chapter, we describe the use of such dyes for interrogating and visualizing ion and voltage gradients in sea urchin embryos.