Stable germline transgenesis using the Minos Tc1/mariner element in the sea urchin Lytechinus pictus.

Stable transgenesis is a transformative tool in model organism biology. Although the sea urchin is one of the oldest animal models in cell and developmental biology, studies in this animal have largely relied on transient manipulation of wild animals, without a strategy for stable transgenesis. Here, we build on recent progress to develop a more genetically tractable sea urchin species, Lytechinus pictus, and establish a robust transgene integration method. Three commonly used transposons (Minos, Tol2 and piggyBac) were tested for non-autonomous transposition, using plasmids containing a polyubiquitin promoter upstream of a H2B-mCerulean nuclear marker. Minos was the only transposable element that resulted in significant expression beyond metamorphosis. F0 animals were raised to sexual maturity, and spawned to determine germline integration and transgene inheritance frequency, and to characterize expression patterns of the transgene in F1 progeny. The results demonstrate transgene transmission through the germline, the first example of a germline transgenic sea urchin and, indeed, of any echinoderm. This milestone paves the way for the generation of diverse transgenic resources that will dramatically enhance the utility, reproducibility and efficiency of sea urchin research.

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.

Localization and origins of juvenile skeletogenic cells in the sea urchin Lytechinuspictus.

The development of the sea urchin larval body plan is well understood from extensive studies of embryonic patterning. However, fewer studies have investigated the late larval stages during which the unique pentaradial adult body plan develops. Previous work on late larval development highlights major tissue changes leading up to metamorphosis, but the location of specific cell types during juvenile development is less understood. Here, we improve on technical limitations by applying highly sensitive hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) to the fast-developing and transparent sea urchin Lytechinus pictus, with a focus on skeletogenic cells. First, we show that HCR-FISH can be used in L. pictus to precisely localize skeletogenic cells in the rudiment. In doing so, we provide a detailed staging scheme for the appearance of skeletogenic cells around the rudiment prior to and during biomineralization and show that many skeletogenic cells unassociated with larval rods localize outside of the rudiment prior to localizing inside. Second, we show that downstream biomineralization genes have similar expression patterns during larval and juvenile skeletogenesis, suggesting some conservation of skeletogenic mechanisms during development between stages. Third, we find co-expression of blastocoelar and skeletogenic cell markers around juvenile skeleton located outside of the rudiment, which is consistent with data showing that cells from the non-skeletogenic mesoderm embryonic lineage contribute to the juvenile skeletogenic cell lineage. This work sets the foundation for subsequent studies of other cell types in the late larva of L. pictus to better understand juvenile body plan development, patterning, and evolution.

An RNA interference approach for functional studies in the sea urchin and its use in analysis of nodal signaling gradients.

Dicer substrate interfering RNAs (DsiRNAs) destroy targeted transcripts using the RNA-Induced Silencing Complex (RISC) through a process called RNA interference (RNAi). This process is ubiquitous among eukaryotes. Here we report the utility of DsiRNA in embryos of the sea urchin Lytechinus variegatus (Lv). Specific knockdowns phenocopy known morpholino and inhibitor knockdowns, and DsiRNA offers a useful alternative to morpholinos. Methods are described for the design of specific DsiRNAs that lead to destruction of targeted mRNA. DsiRNAs directed against pks1, an enzyme necessary for pigment production, show how successful DsiRNA perturbations are monitored by RNA in situ analysis and by qPCR to determine relative destruction of targeted mRNA. DsiRNA-based knockdowns phenocopy morpholino- and drug-based inhibition of nodal and lefty. Other knockdowns demonstrate that the RISC operates early in development as well as on genes that are first transcribed hours after gastrulation is completed. Thus, DsiRNAs effectively mediate destruction of targeted mRNA in the sea urchin embryo. The approach offers significant advantages over other widely used methods in the urchin in terms of cost, and ease of procurement, and offers sizeable experimental advantages in terms of ease of handling, injection, and knockdown validation.

Generation of a homozygous mutant drug transporter (ABCB1) knockout line in the sea urchin Lytechinus pictus.

Sea urchins are premier model organisms for the study of early development. However, the lengthy generation times of commonly used species have precluded application of stable genetic approaches. Here, we use the painted sea urchin Lytechinus pictus to address this limitation and to generate a homozygous mutant sea urchin line. L. pictus has one of the shortest generation times of any currently used sea urchin. We leveraged this advantage to generate a knockout mutant of the sea urchin homolog of the drug transporter ABCB1, a major player in xenobiotic disposition for all animals. Using CRISPR/Cas9, we generated large fragment deletions of ABCB1 and used these readily detected deletions to rapidly genotype and breed mutant animals to homozygosity in the F2 generation. The knockout larvae are produced according to expected Mendelian distribution, exhibit reduced xenobiotic efflux activity and can be grown to maturity. This study represents a major step towards more sophisticated genetic manipulation of the sea urchin and the establishment of reproducible sea urchin animal resources.

Cold storage and cryopreservation methods for spermatozoa of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus.

BACKGROUND: Sea urchins have contributed greatly to knowledge of fertilization, embryogenesis, and cell biology. However, until now, they have not been genetic model organisms because of their long generation times and lack of tools for husbandry and gene manipulation. We recently established the sea urchin Lytechinus pictus, as a multigenerational model Echinoderm, because of its relatively short generation time of 4-6 months and ease of laboratory culture. To take full advantage of this new multigenerational species, methods are needed to biobank and share genetically modified L. pictus sperm. RESULTS: Here, we describe a method, based on sperm ion physiology that maintains L. pictus and Strongylocentrotus purpuratus sperm fertilizable for at least 5-10 weeks when stored at 0°C. We also describe a new method to cryopreserve sperm of both species. Sperm of both species can be frozen and thawed at least twice and still give rise to larvae that undergo metamorphosis. CONCLUSIONS: The simple methods we describe work well for both species, achieving >90% embryo development and producing larvae that undergo metamorphosis to juvenile adults. We hope that these methods will be useful to others working on marine invertebrate sperm.

Developmental single-cell transcriptomics in the Lytechinus variegatus sea urchin embryo.

Using scRNA-seq coupled with computational approaches, we studied transcriptional changes in cell states of sea urchin embryos during development to the larval stage. Eighteen closely spaced time points were taken during the first 24 h of development of Lytechinus variegatus (Lv). Developmental trajectories were constructed using Waddington-OT, a computational approach to 'stitch' together developmental time points. Skeletogenic and primordial germ cell trajectories diverged early in cleavage. Ectodermal progenitors were distinct from other lineages by the 6th cleavage, although a small percentage of ectoderm cells briefly co-expressed endoderm markers that indicated an early ecto-endoderm cell state, likely in cells originating from the equatorial region of the egg. Endomesoderm cells also originated at the 6th cleavage and this state persisted for more than two cleavages, then diverged into distinct endoderm and mesoderm fates asynchronously, with some cells retaining an intermediate specification status until gastrulation. Seventy-nine out of 80 genes (99%) examined, and included in published developmental gene regulatory networks (dGRNs), are present in the Lv-scRNA-seq dataset and are expressed in the correct lineages in which the dGRN circuits operate.

An early global role for Axin is required for correct patterning of the anterior-posterior axis in the sea urchin embryo.

Activation of Wnt/ß-catenin (cWnt) signaling at the future posterior end of early bilaterian embryos is a highly conserved mechanism for establishing the anterior-posterior (AP) axis. Moreover, inhibition of cWnt at the anterior end is required for development of anterior structures in many deuterostome taxa. This phenomenon, which occurs around the time of gastrulation, has been fairly well characterized, but the significance of intracellular inhibition of cWnt signaling in cleavage-stage deuterostome embryos for normal AP patterning is less well understood. To investigate this process in an invertebrate deuterostome, we defined Axin function in early sea urchin embryos. Axin is ubiquitously expressed at relatively high levels in early embryos and functional analysis revealed that Axin suppresses posterior cell fates in anterior blastomeres by blocking ectopic cWnt activation in these cells. Structure-function analysis of sea urchin Axin demonstrated that only its GSK-3ß-binding domain is required for cWnt inhibition. These observations and results in other deuterostomes suggest that Axin plays a crucial conserved role in embryonic AP patterning by preventing cWnt activation in multipotent early blastomeres, thus protecting them from assuming ectopic cell fates.

Functional annotation of a hugely expanded nanos repertoire in Lytechinus variegatus, the green sea urchin.

Nanos genes encode essential RNA-binding proteins involved in germline determination and germline stem cell maintenance. When examining diverse classes of echinoderms, typically three, sometimes four, nanos genes are present. In this analysis, we identify and annotate nine nanos orthologs in the green sea urchin, Lytechinus variegatus (Lv). All nine genes are transcribed and grouped into three distinct classes. Class one includes the germline Nanos, with one member: Nanos2. Class two includes Nanos3-like genes, with significant sequence similarity to Nanos3 in the purple sea urchin, Strongylocentrotus purpuratus (Sp), but with wildly variable expression patterns. The third class includes several previously undescribed nanos zinc-finger genes that may be the result of duplications of Nanos2. All nine nanos transcripts occupy unique genomic loci and are expressed with unique temporal profiles during development. Importantly, here we describe and characterize the unique genomic location, conservation, and phylogeny of the Lv ortholog of the well-studied Sp Nanos2. However, in addition to the conserved germline functioning Nanos2, the green sea urchin appears to be an outlier in the echinoderm phyla with eight additional nanos genes. We hypothesize that this expansion of nanos gene members may be the result of a previously uncharacterized L1-class transposon encoded on the opposite strand of a nanos2 pseudogene present on chromosome 12 in this species. The expansion of nanos genes described here represents intriguing insights into germline specification and nanos evolution in this species of sea urchin.

The evolution of a new cell type was associated with competition for a signaling ligand.

There is presently a very limited understanding of the mechanisms that underlie the evolution of new cell types. The skeleton-forming primary mesenchyme cells (PMCs) of euechinoid sea urchins, derived from the micromeres of the 16-cell embryo, are an example of a recently evolved cell type. All adult echinoderms have a calcite-based endoskeleton, a synapomorphy of the Ambulacraria. Only euechinoids have a micromere-PMC lineage, however, which evolved through the co-option of the adult skeletogenic program into the embryo. During normal development, PMCs alone secrete the embryonic skeleton. Other mesoderm cells, known as blastocoelar cells (BCs), have the potential to produce a skeleton, but a PMC-derived signal ordinarily prevents these cells from expressing a skeletogenic fate and directs them into an alternative developmental pathway. Recently, it was shown that vascular endothelial growth factor (VEGF) signaling plays an important role in PMC differentiation and is part of a conserved program of skeletogenesis among echinoderms. Here, we report that VEGF signaling, acting through ectoderm-derived VEGF3 and its cognate receptor, VEGF receptor (VEGFR)-10-Ig, is also essential for the deployment of the skeletogenic program in BCs. This VEGF-dependent program includes the activation of aristaless-like homeobox 1 (alx1), a conserved transcriptional regulator of skeletogenic specification across echinoderms and an example of a "terminal selector" gene that controls cell identity. We show that PMCs control BC fate by sequestering VEGF3, thereby preventing activation of alx1 and the downstream skeletogenic network in BCs. Our findings provide an example of the regulation of early embryonic cell fates by direct competition for a secreted signaling ligand, a developmental mechanism that has not been widely recognized. Moreover, they reveal that a novel cell type evolved by outcompeting other embryonic cell lineages for an essential signaling ligand that regulates the expression of a gene controlling cell identity.

New techniques for creating parthenogenetic larvae of the sea urchin Lytechinus pictus for gene expression studies.

BACKGROUND: Sea urchins are model organisms for studying the spatial-temporal control of gene activity during development. The Southern California species, Lytechinus pictus, has a sequenced genome and can be raised in the laboratory from egg to egg in 4 to 5 months. RESULTS: Here, we present new techniques for generating parthenogenetic larvae of this species and include a gallery of photomicrographs of morphologically abnormal larvae that could be used for transcriptomic analysis. CONCLUSIONS: Comparison of gene expression in parthenogenotes to larvae produced by fertilization could provide novel insights into gene expression controls contributed by sperm in this important model organism. Knowledge gained from transcriptomics of sea urchin parthenogenotes could contribute to parthenogenetic studies of mammalian embryos.

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.

Microbial Composition and Genes for Key Metabolic Attributes in the Gut Digesta of Sea Urchins Lytechinus variegatus and Strongylocentrotus purpuratus Using Shotgun Metagenomics.

This paper describes the microbial community composition and genes for key metabolic genes, particularly the nitrogen fixation of the mucous-enveloped gut digesta of green (Lytechinus variegatus) and purple (Strongylocentrotus purpuratus) sea urchins by using the shotgun metagenomics approach. Both green and purple urchins showed high relative abundances of Gammaproteobacteria at 30% and 60%, respectively. However, Alphaproteobacteria in the green urchins had higher relative abundances (20%) than the purple urchins (2%). At the genus level, Vibrio was dominant in both green (~9%) and purple (~10%) urchins, whereas Psychromonas was prevalent only in purple urchins (~24%). An enrichment of Roseobacter and Ruegeria was found in the green urchins, whereas purple urchins revealed a higher abundance of Shewanella, Photobacterium, and Bacteroides (q-value < 0.01). Analysis of key metabolic genes at the KEGG-Level-2 categories revealed genes for amino acids (~20%), nucleotides (~5%), cofactors and vitamins (~6%), energy (~5%), carbohydrates (~13%) metabolisms, and an abundance of genes for assimilatory nitrogen reduction pathway in both urchins. Overall, the results from this study revealed the differences in the microbial community and genes designated for the metabolic processes in the nutrient-rich sea urchin gut digesta, suggesting their likely importance to the host and their environment.

Neurogenesis in the sea urchin embryo is initiated uniquely in three domains.

Many marine larvae begin feeding within a day of fertilization, thus requiring rapid development of a nervous system to coordinate feeding activities. Here, we examine the patterning and specification of early neurogenesis in sea urchin embryos. Lineage analysis indicates that neurons arise locally in three regions of the embryo. Perturbation analyses showed that when patterning is disrupted, neurogenesis in the three regions is differentially affected, indicating distinct patterning requirements for each neural domain. Six transcription factors that function during proneural specification were identified and studied in detail. Perturbations of these proneural transcription factors showed that specification occurs differently in each neural domain prior to the Delta-Notch restriction signal. Though gene regulatory network state changes beyond the proneural restriction are largely unresolved, the data here show that the three neural regions already differ from each other significantly early in specification. Future studies that define the larval nervous system in the sea urchin must therefore separately characterize the three populations of neurons that enable the larva to feed, to navigate, and to move food particles through the gut.

Colorless spherule cells and lysozyme contribute to innate immunological responses in the sea urchin Lytechinus variegatus, exposed to bacterial challenge.

The sea urchin Lytechinus variegatus is considered a good candidate for aquaculture, but bacterial diseases are a major challenge in culture conditions. The innate immunological defenses of L. variegatus to bacterial challenges were assessed through hematology parameters, in vitro phagocytosis, lysozyme activity and total plasma protein concentrations in cell-free coelomic fluid. Adult sea urchins were inoculated with Microccocus lysodeikticus, Escherichia coli and Vibrio parahaemolyticus in the cavity coelomic. Filtrated and sterile seawater (FSW) injected and non-injected sea urchins were used as control groups. Righting time, external aspects and behavior of sea urchins were evaluated. Twenty-four hours post-inoculation, we found an increase in the population of colorless spherule cells (CLS), phagocytosis, and humoral responses in sea urchins challenged by bacterial inoculations. Righting time was not affected by the treatments and apparent external signs of disease were not observed at least during 96h post-inoculation. The immunological system of L. variegatus quickly eliminated pathogenic microorganisms. CLS and lysozyme activity cooperate in the immune defenses of L. variegatus, showing an extraordinary efficiency for adjusting the immune defenses under stress caused by microbes. We recommend that the cellular and humoral markers serve as routine tests to monitor health status in sea urchins.

Toposome: Source of antimicrobial molecules in the gonads of the sea urchin Lytechinus variegatus (Lamarck, 1816).

Sea urchins live in a challenging environment that requires rapid and efficient responses against pathogens and invaders. This response may be also important in reproductive processes once males and females release their gametes into water. In addition, the gonads are organs with dual function: reproductive organ and nutrient reserve, therefore it needs efficient protective mechanisms to preserve the nutrients as well as the reproductive cells. The aim of this study was to evaluate the presence and characterize antimicrobial molecules in the male and female gonads of the sea urchin Lytechinus variegatus. Through HPLC purification, antimicrobial activity test and mass spectrometry several antimicrobial molecules were found in the gonads of both gender. Computational in silico analyses showed that they are fragments of a glycoprotein called toposome, also known as major yolk protein (MYP) which is one of the major proteins found in the gonads. Although different functions have been reported for this protein, this is the first description of a direct antimicrobial activity in Lytechinus variegatus. The results indicate that when undergoing proteolysis the toposome generates different fragments with antimicrobial activity which may indicate the importance of a rapid defense response strategy against invading microorganisms in the gonads used by both males and females sea urchins.

Comparative study of coelomocytes from Arbacia lixula and Lythechinus variegatus: Cell characterization and in vivo evidence of the physiological function of vibratile cells.

The knowledge on echinoderm coelomocytes has increased in recent years, but researchers still face a complex problem: how to obtain purified cells. Even flow cytometry being useful to address coelomocytes in suspension, the need for a method able to provide isolated cells is still noteworthy. Here, we use Imaging Flow Cytometry (IFC) to characterize the coelomocytes of two sea urchin species - Arbacia lixula and Lytechinus variegatus - and obtain gates to isolate cell populations. Then, we used these gates to study the physiological response of A. lixula coelomocytes during an induced immune challenge with Escherichia coli. An analysis of area and aspect ratio parameters of the flow cytometer allowed the identification of two main cell populations in the coelomic fluid: circular and elongated cells. A combination of this method with nucleus labeling using propidium iodide allowed the determination of gates containing isolated subpopulations of vibratile cells, red spherulocytes, and two phagocytes subpopulations in both species. We observed that during an induced bacterial immune challenge, A. lixula was able to modulate coelomocyte frequencies, increasing the phagocytes and decreasing red spherulocytes and vibratile cells. These results indicate that vibratile cells and red spherulocytes act by immobilizing and stoping bacterial growth, respectively, cooperating with phagocytes in the immune response. The use of IFC was fundamental not only to identify specific gates for the main coelomic subpopulations but also allowed the investigation on how echinoids modulate their physiological responses during immune challenges. Furthermore, we provide the first experimental evidence about the role of vibratile cells, corroborating its involvement with the immune system.

Glyconectin Cell Adhesion Epitope, ß-d-GlcpNAc3S-(1→3)-α-l-Fucp, Is Involved in Blastulation of Lytechinus pictus Sea Urchin Embryos.

Glycans, as the most peripheral cell surface components, are the primary candidates to mediate the initial steps of cell recognition and adhesion via glycan-glycan binding. This molecular mechanism was quantitatively demonstrated by biochemical and biophysical measurements at the cellular and molecular level for the glyconectin 1 ß-d-GlcpNAc3S-(1→3)-α-l-Fucp glycan structure (GN1). The use of adhesion blocking monoclonal antibody Block 2 that specifically recognize this epitope showed that, besides Porifera, human colon carcinoma also express this structure in the apical glycocalyx. Here we report that Block 2 selectively immune-precipitate a Mr 580 × 103 (g580) acidic non-glycosaminoglycan glycan from the total protein-free glycans of Lytechinus pictus sea urchin hatched blastula embryos. Immuno-fluorescence confocal light microscopy and immunogold electron microscopy localized the GN1 structure in the apical lamina glycocalyx attachments of ectodermal cells microvilli, and in the Golgi complex. Biochemical and immune-chemical analyses showed that the g580 glycan is carrying about 200 copies of the GN1 epitope. This highly polyvalent g580 glycan is one of the major components of the glycocalyx structure, maximally expressed at hatched blastula and gastrula. The involvement of g580 GN1 epitope in hatched blastula cell adhesion was demonstrated by: (1) enhancement of cell aggregation by g580 and sponge g200 glycans, (2) inhibition of cell reaggregation by Block 2, (3) dissociation of microvilli from the apical lamina matrix by the loss of its gel-like structure resulting in a change of the blastula embryonal form and consequent inhibition of gastrulation at saturating concentration of Block 2, and (4) aggregation of beads coated with the immune-purified g580 protein-free glycan. These results, together with the previous atomic force microscopy measurements of GN1 binding strength, indicated that this highly polyvalent and calcium ion dependent glycan-glycan binding can provide the force of 40 nanonewtons per single ectodermal cell association of microvilli with the apical lamina, and conservation of glycocalyx gel-like structure. This force can hold the weight of 160,000 cells in sea water, thus it is sufficient to establish, maintain and preserve blastula form after hatching, and prior to the complete formation of further stabilizing basal lamina.

Embryo, larval, and juvenile staging of Lytechinus pictus from fertilization through sexual maturation.

BACKGROUND: Sea urchin embryos have been used for more than a century in the study of fertilization and early development. However, several of the species used, such as Strongylocentrotus purpuratus, have long generation times making them suboptimal for transgenerational studies. RESULTS: Here, we present an overview of the development of a rapidly developing echinoderm species, Lytechinus pictus, from fertilization through sexual maturation. When grown at room temperature (20°C) embryos complete the first cell cycle in 90 minutes, followed by subsequent cleavages every 45 minutes, leading to hatching at 9 hours postfertilization (hpf). The swimming embryos gastrulate from 12 to 36 hpf and produce the cells which subsequently give rise to the larval skeleton and immunocytes. Larvae begin to feed at 2 days and metamorphose by 3 weeks. Juveniles reach sexual maturity at 4 to 6 months of age, depending on individual growth rate. CONCLUSIONS: This staging scheme lays a foundation for future studies in L. pictus, which share many of the attractive features of other urchins but have the key advantage of rapid development to sexual maturation. This is significant for multigenerational and genetic studies newly enabled by CRISPR-CAS mediated gene editing.

Conformational properties of l-fucose and the tetrasaccharide building block of the sulfated l-fucan from Lytechinus variegatus.

Although the 3D structure of carbohydrates is known to contribute to their biological roles, conformational studies of sugars are challenging because their chains are flexible in solution and consequently the number of 3D structural restraints is limited. Here, we investigate the conformational properties of the tetrasaccharide building block of the Lytechinus variegatus sulfated fucan composed of the following structure [l-Fucp4(SO3-)-α(1-3)-l-Fucp2,4(SO3-)-α(1-3)-l-Fucp2(SO3-)-α(1-3)-l-Fucp2(SO3-)] and the composing monosaccharide unit Fucp, primarily by nuclear magnetic resonance (NMR) experiments performed at very low temperatures and using H2O as the solvent for the sugars rather than using the conventional deuterium oxide. By slowing down the fast chemical exchange rates and forcing the protonation of labile sites, we increased the number of through-space 1H-1H distances that could be measured by NMR spectroscopy. Following this strategy, additional conformational details of the tetrasaccharide and l-Fucp in solution were obtained. Computational molecular dynamics was performed to complement and validate the NMR-based measurements. A model of the NMR-restrained 3D structure is offered for the tetrasaccharide.