Alizarin Red S (AZ) is a dye that is commonly used in histological studies and textiles. We find that AZ is teratogenic to Lytechinus variegatus sea urchin larvae. Here, we demonstrate that embryos exposed to AZ have abnormal skeletal patterning and perturbed migration of skeletogenic mesenchyme cells, along with mild ectodermal abnormalities and reduced neural connectivity. Temporal transcriptomics reveal delayed development and implicate wide-reaching changes in gene expression with AZ treatment. Particle image velocimetry experiments show that AZ treatment perturbs the normal pattern of fluid flow away from the larval mouth and inhibits larval feeding. Finally, we show that AZ promotes a significant elevation of reactive oxygen species (ROS) via catalase inhibition. ROS-mediated effects are expected to be broad, in agreement with the broad effects of AZ detected by transcriptomics. Catalase knockdown and hydrogen peroxide treatment are each sufficient to quantitatively phenocopy AZ-mediated skeletal patterning defects, while catalase overexpression is sufficient to rescue the AZ skeletal phenotype. This study is the first to define the teratogenic consequences of AZ exposure on development, skeletal patterning and biomineralization.

MARVEL proteins, including those of the CMTM gene family, are multi-pass transmembrane proteins that play important roles in vesicular trafficking and cell migration; however, little is understood about their role in development, and their role in skeletal patterning is unexplored. CMTM4 is the only CMTM family member found in the developmental transcriptome of the sea urchin Lytechinus variegatus. Here, we validate that LvCMTM4 is a transmembrane protein and show that perturbation of CMTM4 expression via zygotic morpholino or mRNA injection perturbs skeletal patterning, resulting in loss of secondary skeletal elements and rotational defects. We also demonstrate that normal levels of CMTM4 are required for normal PMC migration and filopodial organization, and that these effects are not due to gross mis-specification of the ectoderm. Finally, we show that CMTM4 is sufficient to mediate mesenchymal cell-cell adhesion. Taken together, these data suggest that CMTM4 controls PMC migration and biomineralization via adhesive regulation during sea urchin skeletogenesis. This is the first discovery of a functionally required adhesive gene in this skeletal patterning system.

Per-and polyfluorinated substances (PFAS) are synthetic compounds used in the production of fluoropolymer coatings found in products such as non-stick pans, clothing, cosmetics, and food packaging. These highly persistent molecules are known as "forever chemicals" since they neither degrade environmentally nor break down enzymatically within biological systems. PFAS compounds readily contaminate water sources, and as a result, certain PFAS molecules have bioaccumulated in exposed species including humans. The purpose of this study was to define the effect of two PFAS molecules, the ostensibly more toxic perfluorooctanoic acid (PFOA) and the more recent, reportedly safer chemical hexafluoropropylene oxide dimer acid (Gen X), on the development of Lytechinus variegatus sea urchin embryos. We examined the effects of PFOA and Gen X on development and patterning using morphological analysis, immunostaining, HCR-FISH, and Particle Image Velocimetry (PIV). The results show that both PFAS compounds are teratogenic to sea urchin embryos. PFOA and Gen X each function at different intervals during development and provoke distinct phenotypic and gene expression outcomes. Despite beliefs that Gen X would be a safer alternative, our findings indicate that Gen X has earlier and more severe effects on endomesoderm and dorsal-ventral axis specification, neural development and function, and pattern formation compared to PFOA. These results illustrate the dangerous teratogenic potential of environmentally accumulating PFAS like Gen X, underscoring the negative ecological implications that accompany continuing commercial and industrial use of PFAS in the absence of remediation strategies.

Alizarin Red S (AZ) is an anthraquinone dye that is commonly used in histological studies and textiles. Exposure to AZ results in morphological perturbations in several species, including rats, frogs, and dogs; however, the mechanisms by which AZ has these effects is largely unexplored, and little is known about its effect on development. We have previously shown that AZ is teratogenic to sea urchin larvae, and that AZ was the only calcium-binding mineralization marker among five tested that perturbed skeletal patterning. Here, we further characterize these defects and demonstrate that embryos exposed to AZ have abnormal skeletal element rotation, branching, and bending. Immunostains and polychrome labeling reveal delayed migration of primary mesenchyme cells and initiation of biomineralization. Although gross ectodermal dorsal-ventral specification, ciliary band restriction, and neuronal specification occur normally in the majority of AZ-treated embryos, we find abnormal patterning and connectivity of the serotonergic neurons. Temporal transcriptomics comparisons confirm delayed development and implicate changes in neuron-related GO terms with AZ treatment. Particle imaging velocimetry experiments show that ciliary beating normally directs fluid flow into the larval mouth, while AZ treatment perturbs the normal pattern of vortices and redirects flow away from the mouth. These changes in fluid flow have functional consequences for larval feeding behaviors in AZ-treated embryos. Finally, we show that the effects of AZ on skeletal patterning are largely due to its inhibition of catalase and subsequent elevation of reactive oxygen species. Specifically, catalase knockdown and transient hydrogen peroxide treatment are each sufficient to phenocopy the hallmark AZ-mediated skeletal patterning defects. This study is the first to define the teratogenic consequences of AZ exposure and show how its effects on catalase activity impact development, skeletal patterning, and biomineralization.

The larval skeleton of the sea urchin Lytechinus variegatus is an ideal model system for studying skeletal patterning; however, our understanding of the etiology of skeletal patterning in sea urchin larvae is limited due to the lack of approaches to live-image skeleton formation. Calcium-binding fluorochromes have been used to study the temporal dynamics of bone growth and healing. To date, only calcein green has been used in sea urchin larvae to fluorescently label the larval skeleton. Here, we optimize labeling protocols for two additional calcium-binding fluorochromes: xylenol orange and calcein blue- and demonstrate that these fluorochromes can be used individually or in nested pulse-chase experiments to understand the temporal dynamics of skeletogenesis and patterning. Using a pulse-chase approach, we show that the initiation of skeletogenesis begins around 15 ​h post fertilization. We also assess the timing of triradiate formation in embryos treated with a range of patterning perturbagens and demonstrate that triradiate formation is delayed and asynchronous in embryos ventralized via treatment with either nickel or chlorate. Finally, we measure the extent of fluorochrome incorporation in triple-labeled embryos to determine the elongation rate of numerous skeletal elements throughout early skeletal patterning and compare this to the rate of skeletal growth in embryos treated with axitinib to inhibit VEGFR. We find that skeletal elements elongate much more slowly in axitinib-treated embryos, and that axitinib treatment is sufficient to induce abnormal orientation of the triradiates.

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 in Lytechinus variegatus sea 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 knockdown, indicating that the spatial expansion of Wnt5 is responsible for the patterning defects induced by VGSC inhibition. These results demonstrate a previously unreported connection between bioelectrical status and the spatial control of patterning cue expression during embryonic pattern formation.

MOTIVATION:The detection of distinct cellular identities is central to the analysis of single-cell RNA sequencing (scRNA-seq) 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 Identify Cell states Across Treatments (ICAT) that employs self-supervised feature weighting and control-guided clustering to accurately resolve cell states across heterogeneous conditions.RESULTS:Using simulated and real datasets, we show ICAT is superior in identifying and resolving cell states compared with 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 scRNA-seq data.AVAILABILITY AND IMPLEMENTATION:https://github.com/BradhamLab/icat.

Ethanol is a known vertebrate teratogen that causes craniofacial defects as a component of fetal alcohol syndrome (FAS). Our results show that sea urchin embryos treated with ethanol similarly show broad skeletal patterning defects, potentially analogous to the defects associated with FAS. The sea urchin larval skeleton is a simple patterning system that involves only two cell types: the primary mesenchymal cells (PMCs) that secrete the calcium carbonate skeleton and the ectodermal cells that provide migratory, positional, and differentiation cues for the PMCs. Perturbations in RA biosynthesis and Hh signaling pathways are thought to be causal for the FAS phenotype in vertebrates. Surprisingly, our results indicate that these pathways are not functionally relevant for the teratogenic effects of ethanol in developing sea urchins. We found that developmental morphology as well as the expression of some ectodermal and PMC genes was delayed by ethanol exposure. Temporal transcriptome analysis revealed significant impacts of ethanol on signaling and metabolic gene expression, and a disruption in the timing of GRN gene expression that includes both delayed and precocious gene expression throughout the specification network. We conclude that the skeletal patterning perturbations in ethanol-treated embryos likely arise from a loss of temporal synchrony within and between the instructive and responsive tissues.

Species retaining ancestral features, such as species called living fossils, are often regarded as less derived than their sister groups, but such discussions are usually based on qualitative enumeration of conserved traits. This approach creates a major barrier, especially when quantifying the degree of phenotypic evolution or degree of derivedness, since it focuses only on commonly shared traits, and newly acquired or lost traits are often overlooked. To provide a potential solution to this problem, especially for inter-species comparison of gene expression profiles, we propose a new method named "derivedness index" to quantify the degree of derivedness. In contrast to the conservation-based approach, which deals with expressions of commonly shared genes among species being compared, the derivedness index also considers those that were potentially lost or duplicated during evolution. By applying our method, we found that the gene expression profiles of penta-radial phases in echinoderm tended to be more highly derived than those of the bilateral phase. However, our results suggest that echinoderms may not have experienced much larger modifications to their developmental systems than chordates, at least at the transcriptomic level. In vertebrates, we found that the mid-embryonic and organogenesis stages were generally less derived than the earlier or later stages, indicating that the conserved phylotypic period is also less derived. We also found genes that potentially explain less derivedness, such as Hox genes. Finally, we highlight technical concerns that may influence the measured transcriptomic derivedness, such as read depth and library preparation protocols, for further improvement of our method through future studies. We anticipate that this index will serve as a quantitative guide in the search for constrained developmental phases or processes.

A Correction to this paper has been published: https://doi.org/10.1038/s42003-021-02005-4

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

Echinoderms are an exceptional group of bilaterians that develop pentameral adult symmetry from a bilaterally symmetric larva. However, the genetic basis in evolution and development of this unique transformation remains to be clarified. Here we report newly sequenced genomes, developmental transcriptomes, and proteomes of diverse echinoderms including the green sea urchin (L. variegatus), a sea cucumber (A. japonicus), and with particular emphasis on a sister group of the earliest-diverged echinoderms, the feather star (A. japonica). We learned that the last common ancestor of echinoderms retained a well-organized Hox cluster reminiscent of the hemichordate, and had gene sets involved in endoskeleton development. Further, unlike in other animal groups, the most conserved developmental stages were not at the body plan establishing phase, and genes normally involved in bilaterality appear to function in pentameric axis development. These results enhance our understanding of the divergence of protostomes and deuterostomes almost 500 Mya. Li et al. investigate the evolution and genetic basis of the adult pentameral body plan in echinoderms using genomic, transcriptomic, and proteomic data. They determine that the last common ancestor of echinoderms contained an organized Hox cluster and endoskeleton genes, and suggest that cooption of bilateral development genes was involved in evolution of the pentameric body plan.

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.

Embryonic development is arguably the most complex process an organism undergoes during its lifetime, and understanding this complexity is best approached with a systems-level perspective. The sea urchin has become a highly valuable model organism for understanding developmental specification, morphogenesis, and evolution. As a non-chordate deuterostome, the sea urchin occupies an important evolutionary niche between protostomes and vertebrates. Lytechinus variegatus (Lv) is an Atlantic species that has been well studied, and which has provided important insights into signal transduction, patterning, and morphogenetic changes during embryonic and larval development. The Pacific species, Strongylocentrotus purpuratus (Sp), is another well-studied sea urchin, particularly for gene regulatory networks (GRNs) and cis-regulatory analyses. A well-annotated genome and transcriptome for Sp are available, but similar resources have not been developed for Lv. Here, we provide an analysis of the Lv transcriptome at 11 timepoints during embryonic and larval development. Temporal analysis suggests that the gene regulatory networks that underlie specification are well-conserved among sea urchin species. We show that the major transitions in variation of embryonic transcription divide the developmental time series into four distinct, temporally sequential phases. Our work shows that sea urchin development occurs via sequential intervals of relatively stable gene expression states that are punctuated by abrupt transitions.

During sea urchin embryogenesis, primary mesenchyme cells (PMCs) follow a stereotypical migratory program, arrange into a primary pattern, then begin to secrete a bilaterally symmetric calcium carbonate skeleton. Recently identified genes are expressed in spatially-restricted domains within the PMC population (Sun & Ettensohn, 2014). To better understand the molecular mechanisms orchestrating PMC positioning, we are characterizing the expression profiles of PMC subset-specific genes. To deconvolve the spatiotemporal expression patterns within PMCs, we detect cell-specific mRNA expression with combined RNA fluorescence in situ hybridization and immunolabeling of PMCs. Subsequent confocal microscopy provides 3D position and expression information for individual PMCs. We extract PMC positions and relative gene expression levels, then model these results using open-source 3D modeling software. This versatile protocol can be extended to other models and systems.

Greenfield, Jackie and Jeanine M. Davis, Collection to Commerce: Western North Carolina Non-Timber Forest Products and Their Markets.Department of Horticultural Science, last updated: May 9, 2003, pp. 1-112.* Janbez, K. H., S.A. Saeed and A.H. Gilani, An Assessment of the potential of Protopine to Inhibit Microsomal Drug Metabolising Enzymes and Prevent Chemical Induced Heptatotoxicity in Rodents, Pharmacological Research, vol. 38, No. 3, 1998, pp. 1-5.* Liu, Hailing, Brett E. Jones, Cynthia Bradham and Mark J Czaja, Increased cytochrome P4502E1 expression sensitizes hepatocytes to c-Jun-mdiated cell death from TNF, Am J Physiol Gastrointest Liver Physiol (Oct. 24, 2001), pp. 1.* Pedersen, Anni Oyan, Danish Pig Production Report, Feb. 16, 1998, pp. 1-14.* Zdarilova, Adela, et al., Food and Chemical Toxicology 46 (2008) 3721-3726.

The sea urchin larval skeleton offers a simple model for formation of developmental patterns. The calcium carbonate skeleton is secreted by primary mesenchyme cells (PMCs) in response to largely unknown patterning cues expressed by the ectoderm. To discover novel ectodermal cues, we performed an unbiased RNA-Seq-based screen and functionally tested candidates; we thereby identified several novel skeletal patterning cues. Among these, we show that SLC26a2/7 is a ventrally expressed sulfate transporter that promotes a ventral accumulation of sulfated proteoglycans, which is required for ventral PMC positioning and skeletal patterning. We show that the effects of SLC perturbation are mimicked by manipulation of either external sulfate levels or proteoglycan sulfation. These results identify novel skeletal patterning genes and demonstrate that ventral proteoglycan sulfation serves as a positional cue for sea urchin skeletal patterning.

Skeletal patterning in the sea urchin embryo requires coordinated signaling between the pattern-dictating ectoderm and the skeletogenic primary mesenchyme cells (PMCs); recent studies have begun to uncover the molecular basis for this process. Using an unbiased RNA-Seq-based screen, we have previously identified the TGF-ß superfamily ligand, LvBMP5-8, as a skeletal patterning gene in Lytechinus variegatus embryos. This result is surprising, since both BMP5-8 and BMP2/4 ligands have been implicated in sea urchin dorsal-ventral (DV) and left-right (LR) axis specification. Here, we demonstrate that zygotic LvBMP5-8 is required for normal skeletal patterning on the left side, as well as for normal PMC positioning during gastrulation. Zygotic LvBMP5-8 is required for expression of the left-side marker soxE, suggesting that LvBMP5-8 is required for left-side specification. Interestingly, we also find that LvBMP5-8 knockdown suppresses serotonergic neurogenesis on the left side. While LvBMP5-8 overexpression is sufficient to dorsalize embryos, we find that zygotic LvBMP5-8 is not required for normal DV specification or development. In addition, ectopic LvBMP5-8 does not dorsalize LvBMP2/4 morphant embryos, indicating that, in the absence of BMP2/4, BMP5-8 is insufficient to specify dorsal. Taken together, our data demonstrate that zygotic LvBMP5-8 signaling is essential for left-side specification, and for normal left-side skeletal and neural patterning, but not for DV specification. Thus, while both BMP2/4 and BMP5-8 regulate LR axis specification, BMP2/4 but not zygotic BMP5-8 regulates DV axis specification in sea urchin embryos.

Skeletal patterning in the sea urchin embryo requires a conversation between the skeletogenic primary mesenchyme cells (PMCs) and the overlyingpattern-dictatingectoderm;however, our understandingof the molecular basis for this process remains incomplete. Here, we show that TGF-β-receptor signaling is required during gastrulation to pattern the anterior skeleton. To block TGF-β signaling, we used SB431542 (SB43), a specific inhibitor of the TGF-β type I receptor Alk4/5/7. Treatment with SB43 during gastrulation blocks anterior PMC positioning and the formation of the anterior skeleton, but does not perturb general ectoderm specification or development. This is the first example of a signaling event required for patterning of a specific part of the skeleton. Alk4/5/7 inhibition does not prevent the formation of a mouth, although SB43-treated plutei display reduced feeding ability, presumably due to the loss of the structural support for the mouth conferred by the anterior skeleton. Both Univin and Nodal are potential ligands for Alk4/5/7; however, Nodal is unilaterally expressed on only the right side, whereas Univin is bilaterally expressed in the ectoderm adjacent to the anterior skeleton during the relevant time period. Our results demonstrate that Univin is both necessary and sufficient for secondary skeletal development in a control background, consistent with the hypothesis that Univin is a relevant Alk4/5/7 ligand for anterior skeletal patterning. Taken together, our data demonstrate that Alk4/5/7 signaling during gastrulation is required to direct PMCs to the oral hood, and suggest that Univin is a relevant ligand for this signaling event.

Skeletal patterning in the sea urchin embryo requires a conversation between the skeletogenic primary mesenchyme cells (PMCs) and the overlyingpattern-dictatingectoderm;however, our understandingof the molecular basis for this process remains incomplete. Here, we show that TGF-β-receptor signaling is required during gastrulation to pattern the anterior skeleton. To block TGF-β signaling, we used SB431542 (SB43), a specific inhibitor of the TGF-β type I receptor Alk4/5/7. Treatment with SB43 during gastrulation blocks anterior PMC positioning and the formation of the anterior skeleton, but does not perturb general ectoderm specification or development. This is the first example of a signaling event required for patterning of a specific part of the skeleton. Alk4/5/7 inhibition does not prevent the formation of a mouth, although SB43-treated plutei display reduced feeding ability, presumably due to the loss of the structural support for the mouth conferred by the anterior skeleton. Both Univin and Nodal are potential ligands for Alk4/5/7; however, Nodal is unilaterally expressed on only the right side, whereas Univin is bilaterally expressed in the ectoderm adjacent to the anterior skeleton during the relevant time period. Our results demonstrate that Univin is both necessary and sufficient for secondary skeletal development in a control background, consistent with the hypothesis that Univin is a relevant Alk4/5/7 ligand for anterior skeletal patterning. Taken together, our data demonstrate that Alk4/5/7 signaling during gastrulation is required to direct PMCs to the oral hood, and suggest that Univin is a relevant ligand for this signaling event.