Developmental gene regulatory network connections predicted by machine learning from gene expression data alone.

Gene regulatory network (GRN) inference can now take advantage of powerful machine learning algorithms to complement traditional experimental methods in building gene networks. However, the dynamical nature of embryonic development-representing the time-dependent interactions between thousands of transcription factors, signaling molecules, and effector genes-is one of the most challenging arenas for GRN prediction. In this work, we show that successful GRN predictions for a developmental network from gene expression data alone can be obtained with the Priors Enriched Absent Knowledge (PEAK) network inference algorithm. PEAK is a noise-robust method that models gene expression dynamics via ordinary differential equations and selects the best network based on information-theoretic criteria coupled with the machine learning algorithm Elastic Net. We test our GRN prediction methodology using two gene expression datasets for the purple sea urchin, Stronglyocentrotus purpuratus, and cross-check our results against existing GRN models that have been constructed and validated by over 30 years of experimental results. Our results find a remarkably high degree of sensitivity in identifying known gene interactions in the network (maximum 81.58%). We also generate novel predictions for interactions that have not yet been described, which provide a resource for researchers to use to further complete the sea urchin GRN. Published ChIPseq data and spatial co-expression analysis further support a subset of the top novel predictions. We conclude that GRN predictions that match known gene interactions can be produced using gene expression data alone from developmental time series experiments.

microRNA-31 regulates skeletogenesis by direct suppression of Eve and Wnt1.

microRNAs (miRNAs) play a critical role in a variety of biological processes, including embryogenesis and the physiological functions of cells. Evolutionarily conserved microRNA-31 (miR-31) has been found to be involved in cancer, bone formation, and lymphatic development. We previously discovered that, in the sea urchin, miR-31 knockdown (KD) embryos have shortened dorsoventral connecting rods, mispatterned skeletogenic primary mesenchyme cells (PMCs) and shifted and expanded Vegf3 expression domain. Vegf3 itself does not contain miR-31 binding sites; however, we identified its upstream regulators Eve and Wnt1 to be directly suppressed by miR-31. Removal of miR-31's suppression of Eve and Wnt1 resulted in skeletal and PMC patterning defects, similar to miR-31 KD phenotypes. Additionally, removal of miR-31's suppression of Eve and Wnt1 results in an expansion and anterior shift in expression of Veg1 ectodermal genes, including Vegf3 in the blastulae. This indicates that miR-31 indirectly regulates Vegf3 expression through directly suppressing Eve and Wnt1. Furthermore, removing miR-31 suppression of Eve is sufficient to cause skeletogenic defects, revealing a novel regulatory role of Eve in skeletogenesis and PMC patterning. Overall, this study provides a proposed molecular mechanism of miR-31's regulation of skeletogenesis and PMC patterning through its cross-regulation of a Wnt signaling ligand and a transcription factor of the endodermal and ectodermal gene regulatory network.

CRISPR-Cas9 editing of non-coding genomic loci as a means of controlling gene expression in the sea urchin.

We seek to manipulate gene function here through CRISPR-Cas9 editing of cis-regulatory sequences, rather than the more typical mutation of coding regions. This approach would minimize secondary effects of cellular responses to nonsense mediated decay pathways or to mutant protein products by premature stops. This strategy also allows for reducing gene activity in cases where a complete gene knockout would result in lethality, and it can be applied to the rapid identification of key regulatory sites essential for gene expression. We tested this strategy here with genes of known function as a proof of concept, and then applied it to examine the upstream genomic region of the germline gene Nanos2 in the sea urchin, Strongylocentrotus purpuratus. We first used CRISPR-Cas9 to target established genomic cis-regulatory regions of the skeletogenic cell transcription factor, Alx1, and the TGF-ß signaling ligand, Nodal, which produce obvious developmental defects when altered in sea urchin embryos. Importantly, mutation of cis-activator sites (Alx1) and cis-repressor sites (Nodal) result in the predicted decreased and increased transcriptional output, respectively. Upon identification of efficient gRNAs by genomic mutations, we then used the same validated gRNAs to target a deadCas9-VP64 transcriptional activator to increase Nodal transcription directly. Finally, we paired these new methodologies with a more traditional, GFP reporter construct approach to further our understanding of the transcriptional regulation of Nanos2, a key gene required for germ cell identity in S. purpuratus. With a series of reporter assays, upstream Cas9-promoter targeted mutagenesis, coupled with qPCR and in situ RNA hybridization, we concluded that the promoter of Nanos2 drives strong mRNA expression in the sea urchin embryo, indicating that its primordial germ cell (PGC)-specific restriction may rely instead on post-transcriptional regulation. Overall, we present a proof-of-principle tool-kit of Cas9-mediated manipulations of promoter regions that should be applicable in most cells and embryos for which CRISPR-Cas9 is employed.

A single cell RNA sequencing resource for early sea urchin development.

Identifying cell states during development from their mRNA profiles provides insight into their gene regulatory network. Here, we leverage the sea urchin embryo for its well-established gene regulatory network to interrogate the embryo using single cell RNA sequencing. We tested eight developmental stages in Strongylocentrotus purpuratus, from the eight-cell stage to late in gastrulation. We used these datasets to parse out 22 major cell states of the embryo, focusing on key transition stages for cell type specification of each germ layer. Subclustering of these major embryonic domains revealed over 50 cell states with distinct transcript profiles. Furthermore, we identified the transcript profile of two cell states expressing germ cell factors, one we conclude represents the primordial germ cells and the other state is transiently present during gastrulation. We hypothesize that these cells of the Veg2 tier of the early embryo represent a lineage that converts to the germ line when the primordial germ cells are deleted. This broad resource will hopefully enable the community to identify other cell states and genes of interest to expose the underpinning of developmental mechanisms.

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.

Effects of three zinc-containing sunscreens on development of purple sea urchin (Strongylocentrotus purpuratus) embryos.

The growing popularity of physical sunscreens will lead to an increased release of ingredients from zinc oxide (ZnO) sunscreens into marine environments. Though zinc (Zn) is a necessary micronutrient in the ocean, greater than natural Zn concentrations may be released into marine environments by use of sunscreens. The extent of the consequences of this addition of Zn to the ocean are not fully understood. We investigated the effects of materials released by ZnO- sunscreens on the development of California purple sea urchin, Strongylocentrotus purpuratus. Embryos incubated in various concentrations of Zn (0.01, 0.05, 0.1, 0.5, and 1 mg/L), the sources of which included zinc-containing compounds: ZnO and zinc sulfate (ZnSO4); and ZnO sunscreens: All Good, Badger, and Raw Elements brands. Based on EC50 values, ZnO-containing sunscreens were slightly, but not significantly, more toxic than ZnO and ZnSO4, suggesting that sunscreens may release additional unknown materials that are detrimental to sea urchin embryo development. All concentrations of Zn-exposure resulted in significant malformations (skeletal abnormality, stage arrest, axis determination disruption), which were identified using light and fluorescence confocal microscopy. The concentration of Zn2+ internalized by the developing embryos correlated positively with the concentration of Zn in seawater. Additionally, exposure to both ZnO sunscreens and ZnO and ZnSO4 at 1 mg/L Zn, significantly increased calcein-AM (CAM) accumulation, indicating decreased multidrug resistant (MDR) transporter activity. This is one of the first studies documenting ZnO-containing sunscreens release high concentrations of Zn that are internalized by and have detrimental effects on aquatic organisms.

A tumor suppressor Retinoblastoma1 is essential for embryonic development in the sea urchin.

BACKGROUND: Embryonic cells and cancer cells share various cellular characteristics important for their functions. It has been thus proposed that similar mechanisms of regulation may be present in these otherwise disparate cell types. RESULTS: To explore how regulative embryonic cells are fundamentally different from cancerous cells, we report here that a fine balance of a tumor suppressor protein Retinoblastoma1 (Rb1) and a germline factor Vasa are important for proper cell proliferation and differentiation of the somatic cells during embryogenesis of the sea urchin. Rb1 knockdown blocked embryonic development and induced Vasa accumulation in the entire embryo, while its overexpression resulted in a smaller-sized embryo with differentiated body structures. These results suggest that a titrated level of Rb1 protein may be essential for a proper balance of cell proliferation and differentiation during development. Vasa knockdown or overexpression, on the other hand, reduced or increased Rb1 protein expression, respectively. CONCLUSIONS: Taken together, it appears that Vasa protein positively regulates Rb1 protein while Rb1 protein negatively regulates Vasa protein, balancing the act of these two antagonistic molecules in somatic cells. This mechanism may provide a fine control of cell proliferation and differentiation, which is essential for regulative embryonic development.

Distinct transcriptional regulation of Nanos2 in the germ line and soma by the Wnt and delta/notch pathways.

Specification of the primordial germ cells (PGCs) is essential for sexually reproducing animals. Although the mechanisms of PGC specification are diverse between organisms, the RNA binding protein Nanos is consistently required in the germ line in all species tested. How Nanos is selectively expressed in the germ line, however, remains largely elusive. We report that in sea urchin embryos, the early expression of Nanos2 in the PGCs requires the maternal Wnt pathway. During gastrulation, however, Nanos2 expression expands into adjacent somatic mesodermal cells and this secondary Nanos expression instead requires Delta/Notch signaling through the forkhead family member FoxY. Each of these transcriptional regulators were tested by chromatin immunoprecipitation analysis and found to directly interact with a DNA locus upstream of Nanos2. Given the conserved importance of Nanos in germ line specification, and the derived character of the micromeres and small micromeres in the sea urchin, we propose that the ancestral mechanism of Nanos2 expression in echinoderms was by induction in mesodermal cells during gastrulation.

An optogenetic approach to control protein localization during embryogenesis of the sea urchin.

Light inducible protein-protein interactions have been used to manipulate protein localization and function in the cell with utmost spatial and temporal precision. In this technical report, we use a recently developed optogenetic approach to manipulate protein localization in the developing sea urchin embryo. A photosensitive LOV domain from Avena sativa phototropin1 cages a small peptide that binds the engineered PDZ domain (ePDZ) upon blue light irradiation. Using this system, mCherry tagged proteins fused with the LOV domain were recruited to ectopic sub-cellular regions such as the membrane, microtubules, or actin by GFP tagged proteins fused with the ePDZ domain upon blue light irradiation within 1-3 min in the sea urchin embryo. The efficiency and speed of recruitment of each protein to its respective subcellular region appeared to be dependent on the power and duration of laser irradiation, as well as the respective level of affinity to the tagged location. Controlled laser irradiation allowed partial recruitment of the spindle to the membrane, and resulted in cell blebbing. Vasa, a cell cycle and germline factor that localizes on the spindle and enriches in the micromeres at 8-16 cell stage was recruited to ectopic sites, preventing normal enrichment. Continuous blue light activation with a regular blue aquarium light over two days of culture successfully induced LOV-ePDZ binding in the developing embryos, resulting in continued ectopic recruitment of Vasa and failure in gastrulation at Day 2. Although some cytotoxicity was observed with prolonged blue light irradiation, this optogenetic system provides a promising approach to test the sub-cellular activities of developmental factors, as well as to alter protein localization and development during embryogenesis.

Single nucleotide editing without DNA cleavage using CRISPR/Cas9-deaminase in the sea urchin embryo.

BACKGROUND: A single base pair mutation in the genome can result in many congenital disorders in humans. The recent gene editing approach using CRISPR/Cas9 has rapidly become a powerful tool to replicate or repair such mutations in the genome. These approaches rely on cleaving DNA, while presenting unexpected risks. RESULTS: In this study, we demonstrate a modified CRISPR/Cas9 system fused to cytosine deaminase (Cas9-DA), which induces a single nucleotide conversion in the genome. Cas9-DA was introduced into sea urchin eggs with sgRNAs targeted for SpAlx1, SpDsh, or SpPks, each of which is critical for skeletogenesis, embryonic axis formation, or pigment formation, respectively. We found that both Cas9 and Cas9-DA edit the genome, and cause predicted phenotypic changes at a similar efficiency. Cas9, however, resulted in significant deletions in the genome centered on the gRNA target sequence, whereas Cas9-DA resulted in single or double nucleotide editing of C to T conversions within the gRNA target sequence. CONCLUSIONS: These results suggest that the Cas9-DA approach may be useful for manipulating gene activity with decreased risks of genomic aberrations. Developmental Dynamics 246:1036-1046, 2017. © 2017 Wiley Periodicals, Inc.

Parallel embryonic transcriptional programs evolve under distinct constraints and may enable morphological conservation amidst adaptation.

Embryonic development evolves by balancing stringent morphological constraints with genetic and environmental variation. The design principle that allows developmental transcriptional programs to conserve embryonic morphology while adapting to environmental changes is still not fully understood. To address this fundamental challenge, we compare developmental transcriptomes of two sea urchin species, Paracentrotus lividus and Strongylocentrotus purpuratus, that shared a common ancestor about 40 million years ago and are geographically distant yet show similar morphology. We find that both developmental and housekeeping genes show highly dynamic and strongly conserved temporal expression patterns. The expression of other gene sets, including homeostasis and response genes, show divergent expression which could result from either evolutionary drift or adaptation to local environmental conditions. The interspecies correlations of developmental gene expressions are highest between morphologically similar developmental time points whereas the interspecies correlations of housekeeping gene expression are high between all the late zygotic time points. Relatedly, the position of the phylotypic stage varies between these two groups of genes: developmental gene expression shows highest conservation at mid-developmental stage, in agreement with the hourglass model while the conservation of housekeeping genes keeps increasing with developmental time. When all genes are combined, the relationship between conservation of gene expression and morphological similarity is partially masked by housekeeping genes and genes with diverged expression. Our study illustrates various transcriptional programs that coexist in the developing embryo and evolve under different constraints. Apparently, morphological constraints underlie the conservation of developmental gene expression while embryonic fitness requires the conservation of housekeeping gene expression and the species-specific adjustments of homeostasis gene expression. The distinct evolutionary forces acting on these transcriptional programs enable the conservation of similar body plans while allowing adaption.

Characterization and expression analysis of Galnts in developing Strongylocentrotus purpuratus embryos.

Mucin-type O-glycosylation is a ubiquitous posttranslational modification in which N-Acetylgalactosamine (GalNAc) is added to the hydroxyl group of select serine or threonine residues of a protein by the family of UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferases (GalNAc-Ts; EC 2.4.1.41). Previous studies demonstrate that O-glycosylation plays essential roles in protein function, cell-cell interactions, cell polarity and differentiation in developing mouse and Drosophila embryos. Although this type of protein modification is highly conserved among higher eukaryotes, little is known about this family of enzymes in echinoderms, basal deuterostome relatives of the chordates. To investigate the potential role of GalNAc-Ts in echinoderms, we have begun the characterization of this enzyme family in the purple sea urchin, S. purpuratus. We have fully or partially cloned a total of 13 genes (SpGalnts) encoding putative sea urchin SpGalNAc-Ts, and have confirmed enzymatic activity of five recombinant proteins. Amino acid alignments revealed high sequence similarity among sea urchin and mammalian glycosyltransferases, suggesting the presence of putative orthologues. Structural models underscored these similarities and helped reconcile some of the substrate preferences observed. Temporal and spatial expression of SpGalnt transcripts, was studied by whole-mount in situ hybridization. We found that many of these genes are transcribed early in developing embryos, often with restricted expression to the endomesodermal region. Multicolor fluorescent in situ hybridization (FISH) demonstrated that transcripts encoding SpGalnt7-2 co-localized with both Endo16 (a gene expressed in the endoderm), and Gcm (a gene expressed in secondary mesenchyme cells) at the early blastula stage, 20 hours post fertilization (hpf). At late blastula stage (28 hpf), SpGalnt7-2 message co-expresses with Gcm, suggesting that it may play a role in secondary mesenchyme development. We also discovered that morpholino-mediated knockdown of SpGalnt13 transcripts, results in a deficiency of embryonic skeleton and neurons, suggesting that mucin-type O-glycans play essential roles during embryonic development in S. purpuratus.

The small GTPase Arf6 regulates sea urchin morphogenesis.

The small GTPase Arf6 is a conserved protein that is expressed in all metazoans. Arf6 remodels cytoskeletal actin and mediates membrane protein trafficking between the plasma membrane in its active form and endosomal compartments in its inactive form. While a rich knowledge exists for the cellular functions of Arf6, relatively little is known about its physiological role in development. This study examines the function of Arf6 in mediating cellular morphogenesis in early development. We dissect the function of Arf6 with a loss-of-function morpholino and constitutively active Arf6-Q67L construct. We focus on the two cell types that undergo active directed migration: the primary mesenchyme cells (PMCs) that give rise to the sea urchin skeleton and endodermal cells that form the gut. Our results indicate that Arf6 plays an important role in skeleton formation and PMC migration, in part due to its ability to remodel actin. We also found that embryos injected with Arf6 morpholino have gastrulation defects and embryos injected with constitutively active Arf6 have endodermal cells detached from the gut epithelium with decreased junctional cadherin staining, indicating that Arf6 may mediate the recycling of cadherin. Thus, Arf6 impacts cells that undergo coordinated movement to form embryonic structures in the developing embryo.

Transcriptional and post-transcriptional regulation of histone variant H2A.Z during sea urchin development.

Histone variant H2A.Z promotes chromatin accessibility at transcriptional regulatory elements and is developmentally regulated in metazoans. We characterize the transcriptional and post-transcriptional regulation of H2A.Z in the purple sea urchin Strongylocentrotus purpuratus. H2A.Z depletion by antisense translation-blocking morpholino oligonucleotides during early development causes developmental collapse, in agreement with its previously demonstrated general role in transcriptional multipotency. During H2A.Z peak expression in 24-h embryos, endogenous H2A.Z 3' UTR sequences stabilize GFP mRNAs relative to those with SV40 3' UTR sequences, although the 3' UTR of H2A.Z does not determine the spatial distribution of H2A.Z transcripts during embryonic and postembryonic development. We elaborated an H2A.Z::GFP BAC reporter that reproduces embryonic H2A.Z expression. Genome-wide chromatin accessibility analysis using ATAC-seq revealed a cis-regulatory module (CRM) that, when deleted, causes a significant decline of the H2A.Z reporter expression. In addition, the mutation of a Sox transcription factor binding site motif and, more strongly, of a Myb motif cause significant decline of reporter gene expression. Our results suggest that an undetermined Myb-family transcription factor controls the transcriptional regulation of H2A.Z.

Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability.

A conserved alternative form of the purple sea urchin HEB/E2-2/E2A transcription factor mediates a switch in E-protein regulatory state in differentiating immune cells.

E-proteins are basic helix-loop-helix (bHLH) transcription factors with essential roles in animal development. In mammals, these are encoded by three loci: E2-2 (ITF-2/ME2/SEF2/TCF4), E2A (TCF3), and HEB (ME1/REB/TCF12). The HEB and E2-2 paralogs are expressed as alternative (Alt) isoforms with distinct N-terminal sequences encoded by unique exons under separate regulatory control. Expression of these alternative transcripts is restricted relative to the longer (Can) forms, suggesting distinct regulatory roles, although the functions of the Alt proteins remain poorly understood. Here, we characterize the single sea urchin E-protein ortholog (SpE-protein). The organization of the SpE-protein gene closely resembles that of the extended HEB/E2-2 vertebrate loci, including a transcript that initiates at a homologous alternative transcription start site (SpE-Alt). The existence of an Alt form in the sea urchin indicates that this feature predates the emergence of the vertebrates. We present additional evidence indicating that this transcript was present in the common bilaterian ancestor. In contrast to the widely expressed canonical form (SpE-Can), SpE-Alt expression is tightly restricted. SpE-Alt is expressed in two phases: first in aboral non-skeletogenic mesenchyme (NSM) cells and then in oral NSM cells preceding their differentiation and ingression into the blastocoel. Derivatives of these cells mediate immune response in the larval stage. Inhibition of SpE-Alt activity interferes with these events. Notably, although the two isoforms are initially co-expressed, as these cells differentiate, SpE-Can is excluded from the SpE-Alt(+) cell population. This mutually exclusive expression is dependent on SpE-Alt function, which reveals a previously undescribed negative regulatory linkage between the two E-protein forms. Collectively, these findings reorient our understanding of the evolution of this transcription factor family and highlight fundamental properties of E-protein biology.

An anterior signaling center patterns and sizes the anterior neuroectoderm of the sea urchin embryo.

Anterior signaling centers help specify and pattern the early anterior neuroectoderm (ANE) in many deuterostomes. In sea urchin the ANE is restricted to the anterior of the late blastula stage embryo, where it forms a simple neural territory comprising several types of neurons as well as the apical tuft. Here, we show that during early development, the sea urchin ANE territory separates into inner and outer regulatory domains that express the cardinal ANE transcriptional regulators FoxQ2 and Six3, respectively. FoxQ2 drives this patterning process, which is required to eliminate six3 expression from the inner domain and activate the expression of Dkk3 and sFRP1/5, two secreted Wnt modulators. Dkk3 and low expression levels of sFRP1/5 act additively to potentiate the Wnt/JNK signaling pathway governing the positioning of the ANE territory around the anterior pole, whereas high expression levels of sFRP1/5 antagonize Wnt/JNK signaling. sFRP1/5 and Dkk3 levels are rigidly maintained via autorepressive and cross-repressive interactions with Wnt signaling components and additional ANE transcription factors. Together, these data support a model in which FoxQ2 initiates an anterior patterning center that implements correct size and positions of ANE structures. Comparisons of functional and expression studies in sea urchin, hemichordate and chordate embryos reveal striking similarities among deuterostome ANE regulatory networks and the molecular mechanism that positions and defines ANE borders. These data strongly support the idea that the sea urchin embryo uses an ancient anterior patterning system that was present in the common ambulacrarian/chordate ancestor.

Cryo-planing of frozen-hydrated samples using cryo triple ion gun milling (CryoTIGM™).

Cryo-SEM is a high throughput technique for imaging biological ultrastructure in its most pristine state, i.e. without chemical fixation, embedding, or drying. Freeze fracture is routinely used to prepare internal surfaces for cryo-SEM imaging. However, the propagation of the fracture plane is highly dependent on sample properties, and the resulting surface frequently shows substantial topography, which can complicate image analysis and interpretation. We have developed a broad ion beam milling technique, called cryogenic triple ion gun milling (CryoTIGM™ ['kri-ə-,tim]), for cryo-planing frozen-hydrated biological specimens. Comparing sample preparation by CryoTIGM™ and freeze fracture in three model systems, Baker's yeast, mouse liver tissue, and whole sea urchin embryos, we find that CryoTIGM™ yields very large (∼700,000 µm(2)) and smooth sections that present ultrastructural details at similar or better quality than freeze-fractured samples. A particular strength of CryoTIGM™ is the ability to section samples with hard-soft contrast such as brittle calcite (CaCO3) spicules in the sea urchin embryo.