Sea Urchin Gastrula Research Articles

Integrative multi-omics increase resolution of the sea urchin posterior gut gene regulatory network at single-cell level.

Drafting gene regulatory networks (GRNs) requires embryological knowledge pertaining to the cell type families, information on the regulatory genes, causal data from gene knockdown experiments and validations of the identified interactions by cis-regulatory analysis. We use multi-omics involving next-generation sequencing to obtain the necessary information for drafting the Strongylocentrotus purpuratus (Sp) posterior gut GRN. Here, we present an update to the GRN using: (1) a single-cell RNA-sequencing-derived cell atlas highlighting the 2 day-post-fertilization (dpf) sea urchin gastrula cell type families, as well as the genes expressed at the single-cell level; (2) a set of putative cis-regulatory modules and transcription factor-binding sites obtained from chromatin accessibility ATAC-seq data; and (3) interactions directionality obtained from differential bulk RNA sequencing following knockdown of the transcription factor Sp-Pdx1, a key regulator of gut patterning in sea urchins. Combining these datasets, we draft the GRN for the hindgut Sp-Pdx1-positive cells in the 2 dpf gastrula embryo. Overall, our data suggest the complex connectivity of the posterior gut GRN and increase the resolution of gene regulatory cascades operating within it.

Thyroid Hormones Accelerate Initiation of Skeletogenesis via MAPK (ERK1/2) in Larval Sea Urchins (Strongylocentrotus purpuratus).

Thyroid hormones are important regulators of development and metabolism in animals. Their function via genomic and non-genomic actions is well-established in vertebrate species but remains largely elusive among invertebrates. Previous work suggests that thyroid hormones, principally 3,5,3′,5′-Tetraiodo-L-thyronine (T4), regulate development to metamorphosis in sea urchins. Here we show that thyroid hormones, including T4, 3,5,3′-triiodo-l-thyronine (T3), and 3,5-Diiodothyronine (T2) accelerate initiation of skeletogenesis in sea urchin gastrulae and pluteus larvae of the sea urchin Strongylocentrotus purpuratus, as measured by skeletal spicule formation. Fluorescently conjugated hormones show T4 binding to primary mesenchyme cells in sea urchin gastrulae. Furthermore, our investigation of TH mediated skeletogenesis shows that Ets1, a transcription factor controlling initiation of skeletogenesis, is a target of activated (phosphorylated) mitogen-activated protein kinase [MAPK; extracellular signal-regulated kinase 1/2 (ERK1/2)]. As well, we show that PD98059, an inhibitor of ERK1/2 MAPK signaling, prevents the T4 mediated acceleration of skeletogenesis and upregulation of Ets1. In contrast, SB203580, an inhibitor of p38 MAPK signaling, did not inhibit the effect of T4. Immunohistochemistry revealed that T4 causes phosphorylation of ERK1/2 in presumptive primary mesenchyme cells and the basal membrane of epithelial cells in the gastrula. Pre-incubation of sea urchin gastrulae with RGD peptide, a competitive inhibitor of TH binding to integrins, inhibited the effect of T4 on skeletogenesis. Together, these experiments provide evidence that T4 acts via a MAPK- (ERK1/2) mediated integrin membrane receptor to accelerate skeletogenesis in sea urchin mesenchyme cells. These findings shed light, for the first time, on a putative non-genomic pathway of TH action in a non-chordate deuterostome and help elucidate the evolutionary history of TH signaling in animals.

Electron microscopic characterization of nuclear egress in the sea urchin gastrula.

Nuclear egress, also referred to as nuclear envelope (NE) budding, is a process of transport in which vesicles containing molecular complexes or viral particles leave the nucleus through budding from the inner nuclear membrane (INM) to enter the perinuclear space. Following this event, the perinuclear vesicles (PNVs) fuse with the outer nuclear membrane (ONM), where they release their contents into the cytoplasm. Nuclear egress is thought to participate in many functions such as viral replication, cellular differentiation, and synaptic development. The molecular basis for nuclear egress is now beginning to be elucidated. Here, we observe in the sea urchin gastrula, using serial section transmission electron microscopy, strikingly abundant PNVs containing as yet unidentified granules that resemble the ribonucleoprotein complexes (RNPs) previously observed in similar types of PNVs. Some PNVs were observed in the process of fusion with the ONM where they appeared to release their contents into the cytoplasm. These vesicles were abundantly observed in all three presumptive germ layers. These findings indicate that nuclear egress is likely to be an important mechanism for nucleocytoplasmic transfer during sea urchin development. The sea urchin may be a useful model to characterize further and gain a better understanding of the process of nuclear egress.

Involvement of l(–)-rhamnose in sea urchin gastrulation: a live embryo assay

The sea urchin embryo is a National Institutes of Health model system that has provided major developments, and is important in human health and disease. To obtain initial insights to identify glycans that mediate cellular interactions, Lytechinus pictus sea urchin embryos were incubated at 24 or 30 h post-fertilization with 0.0009-0.03 M alpha-cyclodextrin, melibiose, L(-)-rhamnose, trehalose, D(+)-xylose or L(-)-xylose in lower-calcium artificial sea water (pH 8.0, 15°C), which speeds the entry of molecules into the interior of the embryos. While α-cyclodextrin killed the embryos, and L(-)-xylose had small effects at one concentration tested, L(-)-rhamnose caused substantially increased numbers of unattached archenterons and exogastrulated embryos at low glycan concentrations after 18-24 h incubation with the sugar. The results were statistically significant compared with the control embryos in the absence of sugar (P < 0.05). The other sugars (melibiose, trehalose, D(+)-xylose) had no statistically significant effects whatsoever at any of the concentrations tested. In total, in the current study, 39,369 embryos were examined. This study is the first demonstration that uses a live embryo assay for a likely role for L(-)-rhamnose in sea urchin gastrula cellular interactions, which have interested investigators for over a century.

Regulation of spblimp1/krox1a, an alternatively transcribed isoform expressed in midgut and hindgut of the sea urchin gastrula

The sea urchin regulatory gene Spblimp1/krox produces alternatively transcribed and spliced isoforms, 1a and 1b, which have different temporal and spatial patterns of expression. Here we describe a cis-regulatory module that controls the expression of the 1a splice form in the midgut and hindgut at the beginning of gastrulation. Conserved sequence patches revealed by a comparison of the blimp1/krox locus in Strongylocentrotus purpuratus and Lytechinus variegatus genomes were tested by gene transfer, in association with GFP or CAT reporter genes. An expression construct containing a conserved sequence patch immediately 5′ of exon 1a included the transcription initiation site for blimp1/krox1a. This construct displays specific mid and hindgut expression, indicating that these are the locations of endogenous blimp1/krox1a transcription during the gastrula stage. Its sequence contains binding sites for Brn1/2/4, Otx, and Blimp1/Krox itself, as predicted in a prior regulatory network analysis.

A novel approach to study adhesion mechanisms by isolation of the interacting system

For decades most investigations into mechanisms of adhesive interactions have examined whole organisms or single cells. Results using whole organisms are often unclear because it may not be known if a probe used in an experiment is directly affecting the cellular interaction under study or if it is an indirect effect resulting from action on some other structure or pathway. Here we develop a novel approach to isolate the structural components of a cellular interaction by dissecting them out of the organism to study them in a pristine environment away from all confounding factors. We used the adhesion between the archenteron and blastocoel roof of the sea urchin gastrula stage embryo as a model that can be replicated in many other developmental and pathological systems. The isolated components of the cellular interaction and those in the whole organism possessed identical cell surface receptors and adhesive affinities.

Appearance of new glycoproteins in methoxychlor-exposed sea urchin gastrulae.

Appearance of new glycoproteins in methoxychlor-exposed sea urchin gastrulae.

Sea Urchin HSF Activity in Vitroand in Transgenic Embryos

Evidence is provided for the presence at the physiological temperature of 20°C of a heat shock transcriptor factor, HSF, in the nuclei of P.lividusembryos. This HSF is able to specifically bind in vitrothe heat shock element, HSE, of the promoter of the hsp70 gene IV, as suggested by DNA–protein binding reactions and DNAse I protection assays. Upon heat-shock, at the temperature of 31°C, its ability to bind the HSE units becomes much higher. The HSF activated by heat-shock drives in vivothe transcription of the β-galactosidase reporter gene in transgenic sea urchin gastrulae. An ATF-like transcription factor, widely described in other organisms but not at all in sea urchins, is also present in the nuclear extracts and is able to bind the consensus individuated in the hsp70 IV gene promoter.

Major Temporal and Spatial Patterns of Gene Expression during Differentiation of the Sea Urchin Embryo

We have investigated the temporal and spatial patterns of accumulation of mRNAs randomly selected from the sea urchin gastrula polyadenylated RNA population. Three different assays show that the predominant temporal pattern of expression, exhibited by about three-fourths of these messages, consists of a large (mean 80-fold) increase in mRNA abundance between egg and gastrula stages. Most mRNAs are present in the maternal population and are detectable on blots as single mature-sized messages; however, a large number of high-molecular-weight, heterodisperse transcripts containing these same sequences also exist in the egg cytoplasm. The majority of gastrula messages are not embryo specific but are present in total adult urchin RNA at concentrations similar to those in embryos. Fine-scale RNA blot analysis indicates that the majority of mRNAs begin to accumulate at very early blastula stages, although there is considerable diversity in the time when these messages reach peak abundance. Most gastrula mRNAs are also spatially regulated during development. The observed distributions can be categorized into three major functional or regulatory classes: (1) Forty percent of mRNAs accumulate in cells which are cycling or preparing for growth. (2) About one-third of the messages accumulate in one or more differentiating cell types. (3) Only slightly more than one-fourth of the messages are present in all cell types throughout development. Most tissue-specific messages are relatively abundant, indicating that the differentiated functions of cells are executed through mRNAs operating at the level of hundreds of copies per cell. In contrast, most rare messages are expressed in most or all cell types, in which they function at only a few copies per cell. All messages which begin to accumulate before hatching blastula stage are initially distributed broadly, and their distribution becomes progressively restricted during embryogenesis. In contrast, all messages which begin to accumulate after the onset of gastrulation accumulate only in discrete subsets of cells. The results presented here illustrate much more extensive temporal regulation of gene expression during sea urchin embryogenesis than previously detected. This is accompanied by spatial regulation of expression of most genes which is itself temporally modulated as the cellular requirements for cell division and differentiation change during development.

Expression of Na+ , K+ -ATPase α-Subunit in Animalized and Vegetalized Embryos of the Sea Urchin, Hemicentrotus pulcherrimus.

A marked increase in the Na+ , K+ -ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na+ , K+ -ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na+ , K+ -ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na+ , K+ -ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8-16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li+ than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na+ , K+ -ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.

Endo16, a lineage-specific protein of the sea urchin embryo, is first expressed just prior to gastrulation.

We have isolated and characterized a new endoderm-specific gene, designated Endo16, from a sea urchin gastrula stage cDNA library. Northern blot analysis and in situ hybridization experiments indicate that this gene is first expressed in the vegetal plate, a group of endodermal and mesenchymal precursor cells that are poised to invaginate in the first movement of gastrulation. Expression becomes progressively restricted to a subset of endodermal cells as development proceeds. To study the Endo16 gene product, a polyclonal antiserum was raised against bacterially expressed Endo16 protein. Indirect immunofluorescence experiments in midgastrula stage embryos reveal that the Endo16 protein is localized to the surface of endoderm and secondary mesenchyme cells. In Western blot experiments, the antiserum detects a small set of high molecular weight proteins ranging from 180 to greater than 300 kDa. Analysis of the nucleotide-derived amino acid sequence from a partial Endo16 cDNA clone reveals a highly repetitive, extremely acidic protein segment that includes the Arg-Gly-Asp (RGD) tripeptide known to be important in cell binding domains of a number of extracellular proteins. Taken together, these data suggest that the Endo16 protein may be an adhesion molecule involved in gastrulation of the sea urchin embryo.

Territorial localization of heat shock mRNA production in sea urchin gastrulae

In situ hybridization experiments with a labeled DNA probe indicate that the ability to respond to heat shock with the production of the mRNA for the 70 kd heat shock protein is segregated into the ectodermal cells already at the gastrula stage or earlier during the embryonic development of Paracentrotus lividus.

A proteoglycan fraction isolated from the EDTA extract of sea urchin ( Hemicentrotus pulcherrimus) gastrulae stimulates reaggeration of dissociated embryonic cells

A sulfated proteoglycan fraction was prepared from mid-gastrulae Hemicentrotus pulcherrimus by chromatographing the EDTA extract of embryos on DEAE-cellulose and then on Sepharose 6B. A peak fraction (with MW > 10 6) of the Sepharose eluates stimulated in a concentration-dependent manner the reaggregation of cells dissociated from Hemicentrotus blastulae by EDTA treatment, the activity being detected at a concentration of as low as 5.8 μg protein per ml or 2.5 μg fucose per ml. The proteoglycan fraction appeared to contain sulfated fucan and glycosaminoglycan chains linked to a common protein. The glycan moiety obtained by digesting the fraction with protease failed to stimulate the cellular reaggregation.

Cell-specific regulation of protein synthesis in the sea urchin gastrula: A two-dimensional electrophoretic study

Protein synthesis in the two cell types of echinoid early gastrulae was analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Epithelial cells and primary mesenchyme cells were isolated from early gastrulae as described by M. A. Harkey and A. H. Whiteley, 1980 ( Wilhelm. Roux's. Arch. 189, 111–112). Newly synthesized proteins were labeled with [ 3H]valine, extracted in SDS buffer, and analyzed electrophoretically. Of the 454 labeled proteins analyzed, 58 incorporated [ 3H]valine at markedly different relative rates in the two cell types, and 69 were labeled exclusively in one or the other cell type. The most rapidly synthesized proteins in gastrula cells constituted a class which exhibited a much higher degree of cell specificity than the total protein population. Several of these rapidly synthesized proteins were analyzed individually. Among those that were synthesized preferentially in primary mesenchyme cells, two low-molecular-weight, acidic proteins, designated PM28 and PM32, accounted for 9–14% of the total protein synthesis in primary mesenchyme cells but were barely detectable in epithelial cells. Those proteins that were synthesized preferentially in the epithelial cells included several low-molecular-weight species, probably histones, and the cytoskeletal proteins, actin and tubulin. These data indicate that the primary mesenchyme and epithelium of the early gastrula differ profoundly with respect to the synthesis of specific proteins.

A specific replication origin in the chromosomal rDNA of Lytechinus variegatus.

Specific replication origins have been well characterized in prokaryotes1,2, and have been identified in eukaryotic extra-chromosomal DNAs3–8. The best candidate, until now, for a specific eukaryotic chromosomal replication origin has been found in yeast9, but the general existence of such origins is challenged10 by the finding that SV40 and polyoma DNA fragments lacking the viral replication origin can replicate after injection into Xenopus eggs. It has thus been suggested that the origins found on extrachromosomal DNAs exist solely to circumvent the cells' strict requirement for chromosomal DNA to replicate once per cell division. Clearly the disparate views can only be reconciled after the replication of specific chromosomal genes has been studied. To this end, we have studied the replication of the ribosomal genes (rDNA) of the sea urchin Lytechinus variegatus. The visualization of replicating rDNA, isolated from rapidly dividing sea urchin gastrula cells, after restriction endonuclease digestion demonstrates that the initiation of replication in these chromosomal genes is sequence-specific, and is most probably confined to a region within the non-transcribed spacer.

Cell adhesion during gastrulation: A new approach

In gastrulating sea urchin embryos, secondary mesenchyme cells at the tip of the advancing archenteron extend long narrow filopodia which probe the inner surface of the blastocoele wall, rejecting some surface contacts before adhering to other cells. After specific cell adhesions are made, contractions of the filopodia pull the leading tip of the archenteron to the opposite wall of the blastocoele with an accompanying elongation of the archenteron. A study was made of the biochemistry and morphology of the specific adhesions of filopodial extensions by injecting a variety of compounds into the blastocoele of living sea urchin gastrulae and observing their effects on filopodia and cell movements. A number of agents (proteases, lectins) caused specific filopodial detachment and subsequent archenteron regression. Fluorescein-conjugated lectins, including concanavalin A (conA) and wheat germ agglutinin (WGA) exhibited marked specificity of cell surface binding to specific regions (primary mesenchyme cells, blastocoele wall, etc.) of the embryo.

Archenteron cells are responsible for the increase in ribosomal RNA synthesis in sea urchin gastrulae

Paracentrotus lividus embryos were continuously labeled with p 32 from hatching blastula to pluteus. The archenteron cells were then separated from the rest of the embryo and the radioactivity accumulated into the ribosomal RNA of the two cell groups measured. The results clearly indicate that the bulk of ribosomal RNA is mainly if not entirely, synthesized in this time interval by the archenteron cells.

Basic Mechanisms of Ovarian Function: Germ Cells

Recent work is reviewed on several molecular aspects of gene expression during oocyte maturation and early development. Several analyses have been carried out on protein synthesis patterns using the high resolution two-dimensional polyacrylamide gel electrophoresis technique. The protein pattern of some 400–500 spots in sea urchin oocytes before and just after fertilization are quite similar. By gastrula, major stage specific protein changes have been noted. In a similar study major protein changes are noted during the meiotic maturation process in mammals. With the use of the single-copy DNA hybridization technique, the quantitative levels of rare mRNA sequence expression have been determined during oogenesis and early development in the sea urchin model. The mature oocyte sequence set of some 37 × 106 nucleotides of information (or potentially 18,500 different proteins) is synthesized during oogenesis and slowly utilized during development. The sea urchin gastrula mRNA is predominantly synthesized by the embryo genome; however, essentially all those gastrula mRNA sequences can also be found in the mature oocyte (maternal) set. It is proposed by Hough-Evans et al. that this maternal sequence set, which is made during oogenesis and both utilized (translated in 10,000–20,000 proteins), and in part, continually synthesized by all stages of the embryo, plays a critical role in the morphogenic formation of a sea urchin embryo.

Basic mechanisms of ovarian function: germ cells

Recent work is reviewed on several molecular aspects of gene expression during oocyte maturation and early development. Several analyses have been carried out on protein synthesis patterns using the high resolution two-dimensional polyacrylamide gel electrophoresis technique. The protein pattern of some 400-500 spots in sea urchin oocytes before and just after fertilization are quite similar. By gastrula, major stage specific protein changes have been noted. In a similar study major protein changes are noted during the meiotic maturation process in mammals. With the use of the single-copy DNA hybridization technique, the quantitative levels of rare mRNA sequence expression have been determined during oogenesis and early development in the sea urchin model. The mature oocyte sequence set of some 37 x 10(6) nucleotides of information (or potentially 18,500 different proteins) is synthesized during oogenesis and slowly utilized during development. The sea urchin gastrula mRNA is predominantly synthesized by the embryo genome; however, essentially all those gastrula mRNA sequences can also be found in the mature oocyte (maternal) set. It is proposed by Hough-Evans et al. that this maternal sequence set, which is made during oogenesis and both utilized (translated in 10,000-20,000 proteins), and in part, continually synthesized by all stages of the embryo, plays a critical role in the morphogenic formation of a sea urchin embryo.

The same amount of DNA is organized in in vitro-assembled nucleosomes irrespective of the origin of the histones

The four histones H2A, H2B, H3 and H4 from calf thymus, CHO and sea urchin gastrula cells were associated by stepwise dialysis from 2 M NaCl with SV40 DNA Form I. The in vitro-assembled chromatins were visualized by electron microscopy and the size of the DNA fragments generated by digestion with DNase II was determined. Irrespective of the origin of the histones, the size of the smallest DNA band generated at early times of digestion was about 190 base pairs, whereas oligomeric DNA bands were multiples of 140 bp. These results support our previous proposal that the four histones H2A, H2B, H3 and H4 are able to organize more than 140 bp of DNA, but do not provide any evidence that the variability of histones H2A and H2B plays a role in the variability of the DNA repeat length of native chromatins.