Voltage-gated sodium channel activity mediates sea urchin larval skeletal patterning through spatial regulation of Wnt5 expression.

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.

Polychrome labeling reveals skeletal triradiate and elongation dynamics and abnormalities in patterning cue-perturbed embryos.

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 â€‹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.

Ethanol exposure perturbs sea urchin development and disrupts developmental timing.

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.

ICAT: a novel algorithm to robustly identify cell states following perturbations in single-cell transcriptomes.

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.

The developmental transcriptome for Lytechinus variegatus exhibits temporally punctuated gene expression changes.

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.

RNA-Seq identifies SPGs as a ventral skeletal patterning cue in sea urchins.

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.

Late Alk4/5/7 signaling is required for anterior skeletal patterning in sea urchin embryos.

Skeletal patterning in the sea urchin embryo requires a conversation between the skeletogenic primary mesenchyme cells (PMCs) and the overlying pattern-dictating ectoderm; however, our understanding of the molecular basis for this process remains incomplete. Here, we show that TGF-ß-receptor signaling is required during gastrulation to pattern the anterior skeleton. To block TGF-ß signaling, we used SB431542 (SB43), a specific inhibitor of the TGF-ß 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.

Zygotic LvBMP5-8 is required for skeletal patterning and for left-right but not dorsal-ventral specification in the sea urchin embryo.

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.

H(+)/K(+) ATPase activity is required for biomineralization in sea urchin embryos.

The bioelectrical signatures associated with regeneration, wound healing, development, and cancer are changes in the polarization state of the cell that persist over long durations, and are mediated by ion channel activity. To identify physiologically relevant bioelectrical changes that occur during normal development of the sea urchin Lytechinus variegatus, we tested a range of ion channel inhibitors, and thereby identified SCH28080, a chemical inhibitor of the H(+)/K(+) ATPase (HKA), as an inhibitor of skeletogenesis. In sea urchin embryos, the primary mesodermal lineage, the PMCs, produce biomineral in response to signals from the ectoderm. However, in SCH28080-treated embryos, aside from randomization of the left-right axis, the ectoderm is normally specified and differentiated, indicating that the block to skeletogenesis observed in SCH28080-treated embryos is PMC-specific. HKA inhibition did not interfere with PMC specification, and was sufficient to block continuing biomineralization when embryos were treated with SCH28080 after the initiation of skeletogenesis, indicating that HKA activity is continuously required during biomineralization. Ion concentrations and voltage potential were abnormal in the PMCs in SCH28080-treated embryos, suggesting that these bioelectrical abnormalities prevent biomineralization. Our results indicate that this effect is due to the inhibition of amorphous calcium carbonate precipitation within PMC vesicles.

Pantropic retroviruses as a transduction tool for sea urchin embryos.

Sea urchins are an important model for experiments at the intersection of development and systems biology, and technical innovations that enhance the utility of this model are of great value. This study explores pantropic retroviruses as a transduction tool for sea urchin embryos, and demonstrates that pantropic retroviruses infect sea urchin embryos with high efficiency and genomically integrate at a copy number of one per cell. We successfully used a self-inactivation strategy to both insert a sea urchin-specific enhancer and disrupt the endogenous viral enhancer. The resulting self-inactivating viruses drive global and persistent gene expression, consistent with genomic integration during the first cell cycle. Together, these data provide substantial proof of principle for transduction technology in sea urchin embryos.

NF-κB is required for cnidocyte development in the sea anemone Nematostella vectensis.

The sea anemone Nematostella vectensis (Nv) is a leading model organism for the phylum Cnidaria, which includes anemones, corals, jellyfishes and hydras. A defining trait across this phylum is the cnidocyte, an ectodermal cell type with a variety of functions including defense, prey capture and environmental sensing. Herein, we show that the Nv-NF-κB transcription factor and its inhibitor Nv-IκB are expressed in a subset of cnidocytes in the body column of juvenile and adult anemones. The size and distribution of the Nv-NF-κB-positive cnidocytes suggest that they are in a subtype known as basitrichous haplonema cnidocytes. Nv-NF-κB is primarily cytoplasmic in cnidocytes in juvenile and adult animals, but is nuclear when first detected in the 30-h post-fertilization embryo. Morpholino-mediated knockdown of Nv-NF-κB expression results in greatly reduced cnidocyte formation in the 5 day-old animal. Taken together, these results indicate that NF-κB plays a key role in the development of the phylum-specific cnidocyte cell type in Nematostella, likely by nuclear Nv-NF-κB-dependent activation of genes required for cnidocyte development.

A computational model for BMP movement in sea urchin embryos.

Bone morphogen proteins (BMPs) are distributed along a dorsal-ventral (DV) gradient in many developing embryos. The spatial distribution of this signaling ligand is critical for correct DV axis specification. In various species, BMP expression is spatially localized, and BMP gradient formation relies on BMP transport, which in turn requires interactions with the extracellular proteins Short gastrulation/Chordin (Chd) and Twisted gastrulation (Tsg). These binding interactions promote BMP movement and concomitantly inhibit BMP signaling. The protease Tolloid (Tld) cleaves Chd, which releases BMP from the complex and permits it to bind the BMP receptor and signal. In sea urchin embryos, BMP is produced in the ventral ectoderm, but signals in the dorsal ectoderm. The transport of BMP from the ventral ectoderm to the dorsal ectoderm in sea urchin embryos is not understood. Therefore, using information from a series of experiments, we adapt the mathematical model of Mizutani et al. (2005) and embed it as the reaction part of a one-dimensional reaction-diffusion model. We use it to study aspects of this transport process in sea urchin embryos. We demonstrate that the receptor-bound BMP concentration exhibits dorsally centered peaks of the same type as those observed experimentally when the ternary transport complex (Chd-Tsg-BMP) forms relatively quickly and BMP receptor binding is relatively slow. Similarly, dorsally centered peaks are created when the diffusivities of BMP, Chd, and Chd-Tsg are relatively low and that of Chd-Tsg-BMP is relatively high, and the model dynamics also suggest that Tld is a principal regulator of the system. At the end of this paper, we briefly compare the observed dynamics in the sea urchin model to a version that applies to the fly embryo, and we find that the same conditions can account for BMP transport in the two types of embryos only if Tld levels are reduced in sea urchin compared to fly.

Chordin is required for neural but not axial development in sea urchin embryos.

The oral-aboral (OA) axis in the sea urchin is specified by the TGFbeta family members Nodal and BMP2/4. Nodal promotes oral specification, whereas BMP2/4, despite being expressed in the oral territory, is required for aboral specification. This study explores the role of Chordin (Chd) during sea urchin embryogenesis. Chd is a secreted BMP inhibitor that plays an important role in axial and neural specification and patterning in Drosophila and vertebrate embryos. In Lytechinus variegatus embryos, Chd and BMP2/4 are functionally antagonistic. Both are expressed in overlapping domains in the oral territory prior to and during gastrulation. Perturbation shows that, surprisingly, Chd is not involved in OA axis specification. Instead, Chd is required both for normal patterning of the ciliary band at the OA boundary and for development of synaptotagmin B-positive (synB) neurons in a manner that is reciprocal with BMP2/4. Chd expression and synB-positive neural development are both downstream from p38 MAPK and Nodal, but not Goosecoid. These data are summarized in a model for synB neural development.

Spatially mapping gene expression in sea urchin primary mesenchyme cells.

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.

p38 MAPK is essential for secondary axis specification and patterning in sea urchin embryos.

Most eggs in the animal kingdom establish a primary, animal-vegetal axis maternally, and specify the remaining two axes during development. In sea urchin embryos, the expression of Nodal on the oral (ventral) side of the embryo is the first known molecular determinant of the oral-aboral axis (the embryonic dorsoventral axis), and is crucial for specification of the oral territory. We show that p38 MAPK acts upstream of Nodal and is required for Nodal expression in the oral territory. p38 is uniformly activated early in development, but, for a short interval at late blastula stage, is asymmetrically inactivated in future aboral nuclei. Experiments show that this transient asymmetry of p38 activation corresponds temporally to both oral specification and the onset of oral Nodal expression. Uniform inhibition of p38 prevents Nodal expression and axis specification, resulting in aboralized embryos. Nodal and its target Gsc each rescue oral-aboral specification and patterning when expressed asymmetrically in p38-inhibited embryos. Thus, our results indicate that p38 is required for oral specification through its promotion of Nodal expression in the oral territory.

Translational control genes in the sea urchin genome.

Sea urchin eggs and early cleavage stage embryos provide an example of regulated gene expression at the level of translation. The availability of the sea urchin genome offers the opportunity to investigate the "translational control" toolkit of this model system. The annotation of the genome reveals that most of the factors implicated in translational control are encoded by nonredundant genes in echinoderm, an advantage for future functional studies. In this paper, we focus on translation factors that have been shown or suggested to play crucial role in cell cycle and development of sea urchin embryos. Addressing the cap-binding translational control, three closely related eIF4E genes (class I, II, III) are present, whereas its repressor 4E-BP and its activator eIF4G are both encoded by one gene. Analysis of the class III eIF4E proteins in various phyla shows an echinoderm-specific amino acid substitution. Furthermore, an interaction site between eIF4G and poly(A)-binding protein is uncovered in the sea urchin eIF4G proteins and is conserved in metazoan evolution. In silico screening of the sea urchin genome has uncovered potential new regulators of eIF4E sharing the common eIF4E recognition motif. Taking together, these data provide new insights regarding the strong requirement of cap-dependent translation following fertilization. The genome analysis gives insights on the complexity of eEF1B structure and motifs of functional relevance, involved in the translational control of gene expression at the level of elongation. Finally, because deregulation of translation process can lead to diseases and tumor formation in humans, the sea urchin orthologs of human genes implicated in human diseases and signaling pathways regulating translation were also discussed.

The sea urchin kinome: a first look.

This paper reports a preliminary in silico analysis of the sea urchin kinome. The predicted protein kinases in the sea urchin genome were identified, annotated and classified, according to both function and kinase domain taxonomy. The results show that the sea urchin kinome, consisting of 353 protein kinases, is closer to the Drosophila kinome (239) than the human kinome (518) with respect to total kinase number. However, the diversity of sea urchin kinases is surprisingly similar to humans, since the urchin kinome is missing only 4 of 186 human subfamilies, while Drosophila lacks 24. Thus, the sea urchin kinome combines the simplicity of a non-duplicated genome with the diversity of function and signaling previously considered to be vertebrate-specific. More than half of the sea urchin kinases are involved with signal transduction, and approximately 88% of the signaling kinases are expressed in the developing embryo. These results support the strength of this nonchordate deuterostome as a pivotal developmental and evolutionary model organism.

Jun kinase modulates tumor necrosis factor-dependent apoptosis in liver cells.

Tumor necrosis factor (TNF) triggers distinct pathways in liver cells through TNF receptor 1 (TNF-R1) via adapter molecules, including the intracellular cascades leading to apoptosis, nuclear factor-kappa B (NF-kappa B), and Jun kinase (JNK) activation. TNF-dependent activation of NF-kappa B induces the transcription of antiapoptotic genes that renders liver cells resistant against TNF-induced apoptosis. In contrast, the role of JNK during TNF-induced apoptosis is less clear, so we studied its role during this process. Hepatoma cells treated with TNF and cycloheximide undergo apoptosis, which is proceeded by a strong activation of JNK. Adenoviral vectors (adv) were tested to block TNF-dependent JNK activation selectively. An adv expressing dominant-negative (dn) TRAF2 inhibited only JNK and not ERK or NF-kappa B activation. However, the effect of inhibiting JNK activation with a dn TAK1 virus was also specific but was stronger than that via dn TRAF2. In further experiments, the inhibitory effect of dn TAK1 on JNK was used to define its role during TNF-dependent apoptosis. Inhibition of JNK by adv dn TAK1 resulted in an earlier and stronger induction of apoptosis. Interestingly, TAM67, a dn form of c-Jun, did not mediate the JNK-dependent effect on TNF-dependent apoptosis, indicating that other molecular targets are essential to confer this mechanism. However, the modified apoptosis pattern could be inhibited by adv expressing Bcl-2 or dn FADD. In conclusion, we define TAK1 as a kinase specifically involved in TNF-induced JNK activation in hepatoma cells and show that JNK transduces antiapoptotic signals, which modulate the strength and time course of FADD-dependent cell death involving mitochondrial permeability transfer.