Mesenchyme Blastula Research Articles

Revealing the cells fated to form the cell mass in embryos of temnopleurid sea urchins

Larvae of temnopleurid sea urchins form a cell mass (CM) instead of an amniotic cavity on the left side at the early developmental stage for formation of the adult rudiment. However, the cell lineage and the mechanisms that form the CM are still unknown. We analyzed the potential to form a CM in partial embryos resulting from microsurgeries, using two temnopleurid species, Mespilia globulus (L.) and Temnopleurus toreumaticus (Leske). CM formation was completed 28-34 h after fertilization at 24°C, corresponding to the period from the late prism to the two-armed pluteus larval stages in both species. In the case of specimens dissected horizontally during the mesenchyme blastula to prism stages, the CM was formed in partial embryos containing enough of the an2 region, a part of the precursor cells that differentiate the ectoderm. The proportion of specimens with a CM was higher in T. toreumaticus than in M. globulus. Additionally, all larvae derived from half embryos obtained from dissection along the animal-vegetal axis at the mesenchyme blastula stage formed the CM. Transplantation of a stained animal or vegetal hemisphere at the 16-cell stage into a nonstained vegetal or animal embryo indicated that the CM derives from the animal half. Exogastrulae vegetalized by lithium chloride treatment did not form the CM. These results indicate that the CM formation is dependent not only on the an2 region but also on signals from the vegetal region after the mesenchyme blastula stage.

The ontogeny of synaptophysin expression patterns on the GABAergic ciliary band-associated strand during larval development of the sea urchin, Hemicentrotus pulcherrimus A. Agassiz, 1864

The swimming activity of sea urchin larvae depends on the ciliary beating primarily generated at the circumoral ciliary band (CB) and is regulated by several neurotransmitters, including γ-aminobutyric acid (GABA). Although GABA is primarily localized in the CB, its synthetase glutamate decarboxylase (GAD) is not expressed in the CB but in the ciliary band-associated strand (CBAS). The CBAS expresses synaptophysin (Syp), a major synaptic vesicle component that is detected in the GAD-expressing puncta. In this study, we analyzed the ontogeny of the spatiotemporal expression pattern of Syp. Syp was initially detected in the cytoplasm of small patches of ectodermal cells from the mesenchyme blastula stage, then along with GABA from the mid-gastrula stage. At the prism stage, the blastocoelar cells also expressed Syp and GABA. In larvae, GABA was detected in the CBAS and the CB. The latter also expressed the GABA(A) receptor. A GAD inhibitor, 3-mercaptopropionic acid, inhibited GABA expression in the CBAS and the CB. During and after the 4-arm pluteus stage (4aPL), the CBAS completed the encircling of the oral ectoderm region, which, however, left the CBAS-absent upper oral lobe region. The present study indicated close localization of GABA and Syp in the puncta of the CBAS and that of GABA and GABA(A)R in the CB, and that, for the first time in the sea urchin nervous system, implicates the Syp-mediated efferent GABA transmission from the CBAS to the CB.

An empirical model of Onecut binding activity at the sea urchin SM50 C-element gene regulatory region

Studying the formation of endoskeleton in many species is complex and difficult. The sea urchin embryo offers an unparalleled platform for understanding this process because of the ease with which its skeletogenic mesenchyme cells can be manipulated. In this study, preliminary evidence from biochemical studies towards understanding the role of the Onecut transcription factor during sea urchin skeletogenic mesenchyme cell specification is presented. Based on the evidence, an empirical model is proposed showing how Onecut, together with associated co-factors, may be using the C-element of the SM50 gene regulatory region in advance of the sea urchin Strongylocentrotus purpuratus spicule development. In the model, Onecut recognizes and binds the DNA sequence CATCGATCTC in the C-element without temporal restriction. Onecut then utilizes different sets of co-factors to switch from its unknown function early in development (four cell stage to the mesenchyme blastula stage), to its known role in the oral-aboral boundary thereafter. At the writing of this report, definitive evidence as to whether the "early" factors are expressed in all cells except the micromere lineages, or whether the "late" factors are expressed in micromere descendants or ectodermal precursors only are lacking. The former would suggest a possible Onecut repression function for the early co-factors outside the micromere lineages; the latter scenario would suggest a Onecut activation function for the late co-factors in the presumptive ciliary band.

Characterization and Expression of Conserved Skeletal Proteins in Echinoderms

Specification of the cell fate of skeleton‐forming cells in developing sea urchin embryos has been well characterized and the skeletal proteome of both adult and larval skeleton has been determined. Little is known, however, of the mechanism and proteins involved in the mineralization process. A number of spicule matrix proteins have been identified in sea urchins and are present in the organic matrix during spicule formation. However, loss of function studies have indicated they may not be required for mineralization. We have taken a comparative, evolutionary approach to try and elucidate what proteins may be required for the process of mineralization. Here we report the characterization of the skeletal proteome of the sea star Patiria miniata. Our hypothesis is that there are proteins utilized in skeleton formation that are conserved between all echinoderm groups, and possibly among all deuterostomes. First we have isolated skeletal proteins from P. miniata, and then used LC/MS/MS to obtain peptide sequences of those proteins. Characterization of skeletal proteins was accomplished utilizing the NCBI non‐redundant protein database. By comparing the proteins present in all mineralized tissues of the sea urchin with those found in other echinoderm groups we have identified a set of conserved proteins containing similar functional domains. The skeletal proteome from the two echinoderms, P. miniata and Strongylocentrotus purpuratus, groups also shared novel proteins with similar functional domains. We have analyzed the sequences of the proteins we identified as potentially important to determine the evolutionary relationship and to identify the sequences most highly conserved. Additionally, some of these proteins are also found in vertebrate bone. We have also shown that some skeletal proteins prevalent in one group are not found in other echinoderms. This suggests independent co‐option of proteins utilized in mineralization in some groups. The conserved proteins were then analyzed for temporal expression at eight different stages of the developing embryo of S. purpuratus. Some of these stages, such as mesenchyme blastula, were predicted to show higher level of expression for some of the conserved skeletal proteins. Utilizing gel electrophoresis and quantitative polymerase chain reaction (qPCR), we analyzed the temporal expression of conserved skeletal proteins and the relative expression of those proteins during the eight stages of the developing embryo. We found that expression of the conserved skeletal proteins varied, but all were present when skeleton formation occurred in the embryo.Support or Funding InformationNIH/NIGMS T34GM008074, NSF grant 09097979

Restricted expression of karyopherin alpha mRNA in the sea urchin suggests a role in neurogenesis

Karyopherin alpha (KAP-α) proteins are critical for the transport of many molecules into the nucleus. In this study, we identified three members of the KAP-α family in the sea urchin Lytechinus variegatus and described the developmental expression of these proteins. Although many importins are assumed to have ubiquitous expression, we found that all three genes were differentially expressed. Both LvKPNA1/5/6 and LvKPNA3/4 accumulated at high levels during cleavage, exhibiting cyclic expression as cells divided. By the blastula and gastrula stages expression decreased, remaining highest in the vegetal plate and archenteron, and by the prism/pluteus stages expression was restricted to the oral surface and gut. Expression of a third KAP-α gene, LvKPNA2/7, was examined in embryos from the mesenchyme blastula to pluteus stages. LvKPNA2/7 mRNA is present in vegetal cells of the mesenchyme blastula and, during gastrulation, it is localized to the archenteron and appears in additional groups of ectodermal cells. Prism/pluteus stage embryos expressed LvKPNA2/7 in the gut and scattered distribution of transcripts in the ciliary band resembled expression patterns of neural cells. We hypothesize that LvKPNA2/7 maintains pluripotency in the neural precursors prior to activation of neural differentiation and believe that this study is an important first step in an effort to better understand the roles of importins during embryogenesis.

Glutathione transferase theta in apical ciliary tuft regulates mechanical reception and swimming behavior of Sea Urchin Embryos

An apical tuft, which is observed in a wide range of embryos/larvae of marine invertebrates, is composed of a group of cilia that are longer and less motile than the abundant lateral cilia covering the rest of the embryonic surface. Although the apical tuft has been thought to function as a sensory organ, its molecular composition and roles are poorly understood. Here, we identified a glutathione transferase theta (GSTT) as an abundant and specific component of the apical tuft in sea urchin embryos. The expression of GSTT mRNA increases and becomes limited to the animal plate of the mesenchyme blastula, gastrula, and prism larva. Electron microscopy and tandem mass spectrometry demonstrated that the apical tuft contains almost every axonemal component for ciliary motility. Low concentrations of an inhibitor of glutathione transferase bromosulphophthalein (BSP) induce bending of apical tuft, suggesting that GSTT regulates motility of apical tuft cilia. Embryos treated with BSP swim with normal velocity and trajectories but show less efficiency of changing direction when they collide with an object. These results suggest that GSTT in the apical tuft plays an important role in the mechanical reception for the motility regulation of lateral motile cilia in sea urchin embryos.

Tissue-specificity and phylogenetics of Pl-MT mRNA during Paracentrotus lividus embryogenesis

Metallothioneins (MTs) constitute a family of cysteine-rich, low molecular weight proteins, which generally provide protection against metal toxicity and oxidative stress counteracting the cell damage caused by essential and non-essential heavy metals. Equally important is the physiological role of MTs in the homeostasis of essential metals, which are involved in a wide variety of cellular processes. The aim of this work was to investigate the expression and the territorial localization of Paracentrotus lividus MT (Pl-MT) mRNA during sea urchin development by Quantitative Polymerase Chain Reaction (QPCR) and Whole Mount In Situ Hybridization (WMISH), as well as the phylogenetic comparison with selected MT homologs present in different phyla. We found that Pl-MT mRNA is accumulated in unfertilized eggs and constitutively expressed during development, with very low levels of maternal mRNA at cleavage stages, followed by a significant rise during gastrulation with a peak at the prism stage. Pl-MT mRNA was expressed in the vegetative plate at mesenchyme blastula, later restricted to the endoderm of gastrula embryos and finally to the gut of plutei. Indirect immunofluorescence (IF) using a specific antibody for the endoderm marker Endo1 demonstrated a co-localization with the Pl-MT transcripts in the midgut and hindgut after the intestine differentiation occurs and when larval feeding begins. Our results show for the first time the constitutive temporal and tissue-specific expression of MT in P. lividus embryos, providing new information for studies on the mechanisms controlling basal and induced MT gene expression.The analysis of the phylogenetic relationship of Pl-MT with homologs from different phyla, ranging from yeast to vertebrates, suggests the evolutionary process of these proteins, which could have been selected not only on the basis of their ability to bind metals but also by their tissue-specificity.

Unc-5/netrin-mediated axonal projection during larval serotonergic nervous system formation in the sea urchin, Hemicentrotus pulcherrimus

The molecular structure and role of two splice-isoforms of Unc-5 (Hp-Unc-5v1 and v2) in Unc-5/netrin interaction during serotonergic axonal projection were elucidated in this study. Hp-Unc-5v1 was found to be comprised of two immunoglobulin-like domains, two thrombospondin domains in the extracellular region, and ZU-5, DB, and Death domains in the cytoplasmic region, whereas Hp-Unc-5v2 lacked one thrombospondin domain, the transmembrane domain, and all cytoplasmic domains. Hp-Unc-5v1 was transcribed in unfertilized eggs, which continued until the 3-day post-fertilization (-dpf) 2-arm pluteus stage, but was suspended at the mesenchyme blastula stage (mB1), whereas Hp-Unc-5v2 was not transcribed in unfertilized eggs, but was from after fertilization to the same developmental stage of mB1 as Hp-Unc-5v1. Relative accumulation of transcripts of both splice-isoforms peaked at the prism stage and declined thereafter, and they were localized at the vegetal pole region of early gastrulae, around the blastopore in mid- to late gastrulae, at fore- and mid-gut regions and on the basal side of dorsal ectoderm in 28-hour post-fertilization prism larvae, and within axons at and after the 2-dpf pluteus stage. Hp-Unc-5v2:GFP was detected in the entire serotonergic cell body and extracellularly on the basal surface of oral ectoderm in 2-dpf plutei and exclusively within axons in 4-dpf plutei. Overexpression of Hp-Unc-5v2 resulted in decreased axonal projection in plutei. Knockdown of Hp-Unc-5v1 by morpholino antisense oligonucleotide resulted in severe deficiency of axonal projection. Interference of Unc-5/netrin interaction using an exogenous synthetic SQDFGKTW peptide from the VI domain in Hp-netrin inhibited axonal projection and larval swimming.

Cis-regulatory logic driving glial cells missing: Self-sustaining circuitry in later embryogenesis

The glial cells missing (gcm) regulatory gene of the sea urchin Strongylocentrotus purpuratus is first expressed in veg2 daughter cells as the genomic target of late cleavage stage Delta-Notch signaling from the skeletogenic mesoderm precursors. Gcm is required in veg2 progeny during late cleavages for the early phase of pigment cell precursor specification. Here we report on a later acting cis-regulatory module that assumes control of gcm expression by the early mesenchyme blastula stage and maintains it through pigment cell differentiation and dispersal. Cis-perturbation analyses reveal that the two critical elements within this late module are consensus matches to Gcm and Six1 binding sites. Significantly, six1 mRNA localizes to gcm+cells from the mesenchyme blastula stage onwards. Trans-perturbations with anti-sense morpholinos reveal a co-dependency between six1 and gcm. Six1 mRNA levels fall sharply after Gcm is depleted, while depleting Six1 leads to significant reductions in output of endogenous gcm or modular-reporters. These results support the conclusion gcm and six1 comprise a positive intergenic feedback loop in the mesodermal GRN. This often employed cross regulatory GRN feature here ensures self-sustaining gcm output in a cohort of fully specified pigment cell precursors at a relatively early developmental stage.

The control offoxN2/3expression in sea urchin embryos and its function in the skeletogenic gene regulatory network

Early development requires well-organized temporal and spatial regulation of transcription factors that are assembled into gene regulatory networks (GRNs). In the sea urchin, an endomesoderm GRN model explains much of the specification in the endoderm and mesoderm prior to gastrulation, yet some GRN connections remain incomplete. Here, we characterize FoxN2/3 in the primary mesenchyme cell (PMC) GRN state. Expression of foxN2/3 mRNA begins in micromeres at the hatched blastula stage and then is lost from micromeres at the mesenchyme blastula stage. foxN2/3 expression then shifts to the non-skeletogenic mesoderm and, later, to the endoderm. Here, we show that Pmar1, Ets1 and Tbr are necessary for activation of foxN2/3 in micromeres. The later endomesoderm expression of foxN2/3 is independent of the earlier expression of foxN2/3 in micromeres and is independent of signals from PMCs. FoxN2/3 is necessary for several steps in the formation of the larval skeleton. Early expression of genes for the skeletal matrix is dependent on FoxN2/3, but only until the mesenchyme blastula stage as foxN2/3 mRNA disappears from PMCs at that time and we assume that the protein is not abnormally long-lived. Knockdown of FoxN2/3 inhibits normal PMC ingression and foxN2/3 morphant PMCs do not organize in the blastocoel and fail to join the PMC syncytium. In addition, without FoxN2/3, the PMCs fail to repress the transfating of other mesodermal cells into the skeletogenic lineage. Thus, FoxN2/3 is necessary for normal ingression, for expression of several skeletal matrix genes, for preventing transfating and for fusion of the PMC syncytium.

Involvement of Delta and Nodal signals in the specification process of five types of secondary mesenchyme cells in embryo of the sea urchin, Hemicentrotus pulcherrimus

Secondary mesenchyme cells (SMCs) of the sea urchin embryo are composed of pigment cells, blastocoelar cells, spicule tip cells, coelomic pouch cells and muscle cells. To learn how and when these five types of SMCs are specified in the veg₂ descendants, Notch or Nodal signaling was blocked with γ-secretase inhibitor or Nodal receptor inhibitor, respectively. All types of SMCs were decreased with DAPT, while sensitivity to this inhibitor varied among them. Pulse-treatment revealed that five types of SMCs are divided into "early" (pigment cells and blastocoelar cells) and "late" (spicule tip cells, coelomic pouch cells and muscle cells) groups; the "early" group was sensitive to DAPT up to the hatching, and the "late" group was sensitive until the mesenchyme blastula stage. Judging from timing of the shift of Delta-expressing regions, it was suggested that the "early" group and "late" groups are derived from the lower and the middle tier of veg₂ descendants, respectively. Interestingly, numbers of SMCs were also altered with SB431542; blastocoelar cells, coelomic pouch cells and circum-esophageal muscles decreased, whereas pigment cells and spicule tip cells increased in number. Pulse-treatment showed that the "early" group was sensitive up to the mesenchyme blastula stage, while the "late" group up to the onset of gastrulation. Thus, it became clear that precursor cells of the "early" and "late" groups, which are located in different regions in the vegetal plate, receive Delta and Nodal signals at different timings, resulting in the diversification of SMCs. Based on the obtained results, the specification processes of five types of SMCs are diagrammatically presented.

The dynamic gene expression patterns of transcription factors constituting the sea urchin aboral ectoderm gene regulatory network

The temporal and spatial expression patterns of regulatory genes are required for building a gene regulatory network (GRN). The current ectoderm GRN model for the sea urchin embryo includes pregastrular specification functions in the oral (OE) and aboral ectoderm (AE). Unlike the OE, which is resolved into several subdomains, the AE is considered a simpler territory due to the lack of detailed gene expression studies in this territory. Here, we perform temporal and spatial gene expression studies on the eight transcription factor genes constituting the AE GRN. Based on the differential gene expression patterns, we conclude that the AE contains at least three subdomains at the mesenchyme blastula stage. We also performed immunostaining for pSmad1/5/8 to monitor the activation of the BMP signaling pathway. The dynamic changes in the expression patterns of these transcription factor genes and the nuclearization of pSmad1/5/8 may provide a foundation for resolving the AE GRN.

AnkAT-1 is a novel gene mediating the apical tuft formation in the sea urchin embryo

In sea urchin embryos, the apical tuft forms within the neurogenic animal plate. When FoxQ2, one of the earliest factors expressed specifically in the animal plate by early blastula stage, is knocked down, the structure of the apical tuft is altered. To determine the basis of this phenotype, we identified FoxQ2-dependent genes using microarray analysis. The most strongly down-regulated gene in FoxQ2 morphants encodes a protein with ankyrin repeats region in its N-terminal domain. We named this gene ankAT-1, Ankyrin-containing gene specific for Apical Tuft. Initially its expression in the animal pole region of very early blastula stage embryos is FoxQ2-independent but becomes FoxQ2-dependent beginning at mesenchyme blastula stage and continuing in the animal plate of 3-day larvae. Furthermore, like FoxQ2, this gene is expressed throughout the expanded apical tuft region that forms in embryos lacking nuclear β-catenin. When AnkAT-1 is knocked-down by injecting a morpholino, the cilia at the animal plate in the resulting embryos are much shorter and their motility is less than that of motile cilia in other ectoderm cells, and remains similar to that of long apical tuft cilia. We conclude that AnkAT-1 is involved in regulating the length of apical tuft cilia.

Transcriptional increase and misexpression of 14-3-3 epsilon in sea urchin embryos exposed to UV-B

Members of the 14-3-3 protein family are involved in many important cellular events, including stress response, survival and apoptosis. Genes of the 14-3-3 family are conserved from plants to humans, and some members are responsive to UV radiation. Here, we report the isolation of the complete cDNA encoding the 14-3-3 epsilon isoform from Paracentrotus lividus sea urchin embryos, referred to as Pl14-3-3ε, and the phylogenetic relationship with other homologues described in different phyla. Pl14-3-3ε mRNA levels were measured by QPCR during development and found to increase from the mesenchyme blastula to the prism stage. In response to UV-B (312nm) exposure, early stage embryos collected 2h later showed a 2.3-fold (at 400J/m(2)) and a 2.7-fold (at 800J/m(2)) increase in Pl14-3-3ε transcript levels compared with controls. The spatial expression of Pl14-3-3ε mRNA, detected by whole mount in situ hybridization in both control and UV-B exposed embryos, harvested at late developmental stages, showed transcripts to be located in the archenteron of gastrula stage and widely distributed in all germ layers, respectively. The Pl14-3-3ε mRNA delocalization parallels the failure in archenteron elongation observed morphologically, as well as the lack of specific endoderm markers, investigated by indirect immuno-fluorescence on whole mount embryos. Results confirm the involvement of 14-3-3ε in the stress response elicited by UV-B and demonstrate, for the first time, its contribution at the transcriptional level in the sea urchin embryo.

Dicer is Required for the Normal Development of Sea Urchin,Hemicentrotus pulcherrimus

MicroRNAs are single-stranded RNA molecules with a length of 19-25 nucleotides, which play roles in various biological phenomena, including development, differentiation, apoptosis, by regulating target gene expression. Although the presence of microRNA molecules in sea urchin and the expression of genes involved in microRNA biogenesis during sea urchin development have been reported recently, the function of microRNA in sea urchin development remains to be elucidated. In this study, to understand the function of microRNA in the early development of sea urchin, we focused on Dicer, an essential enzyme for biosynthesis of mature microRNA. We determined the nucleotide sequence of cDNA for a Dicer homolog in the sea urchin, Hemicentrotus pulcherrimus, HpDcr, and found that functional domains of Dicer proteins are conserved in HpDcr. Analyses of its pattern of expression showed that HpDcr mRNA is expressed in embryos at all developmental stages analyzed, and seems to distribute asymmetrically at the morula and later stages. Knockdown of HpDcr resulted in anomalous morphogenesis, such as impairment of gastrulation and skeletogenesis at the mesenchyme blastula stage and later stages, and alteration of mRNA levels of cell type-specific genes. Thus, HpDcr plays important roles in morphogenesis in sea urchin embryos, suggesting that miRNA could be involved in the early development of sea urchin by regulating target gene expression.

Genetic Hypervariability in Two Distinct Deuterostome Telomerase Reverse Transcriptase Genes and their Early Embryonic Functions

Functional proteins of complex eukaryotes within the same species are rather invariant. A single catalytic component of telomerase TERT is essential for an active telomerase complex that maintains telomeres. Surprisingly, we have identified two paralogous SpTERT-L and SpTERT-S genes with novel domains in Strongylocentrotus purpuratus (purple sea urchin). The SpTERT-S and SpTERT-L genes were differentially expressed throughout embryogenesis. An unusual germline nucleotide substitution and amino acid variation was evident in these TERTs. The hypervariability of SpTERT-S haplotypes among different individuals reached unprecedented levels of pi > 0.2 in exon 11 region. The majority of nucleotide changes observed led to nonsynonymous substitutions creating novel amino acids and motifs, suggesting unusual positive selection and rapid evolution. The majority of these variations were in domains involved in binding of SpTERT to its RNA component. Despite hypervariability at protein level, SpTERT-S conferred telomerase activity, and its suppression during early embryogenesis led to arrest at late mesenchymal blastula. Domain exchange and embryo rescue experiments suggested that SpTERT may have evolved functions unrelated to classic telomerase activity. We suggest that telomerase has a specific and direct function that is essential for integration of early polarity signals that lead to gastrulation. Identification of these unique hypervariable telomerases also suggests presence of a diversity generation mechanism that inculcates hypervariable telomerases and telomere lengths in germline.

Specification process of animal plate in the sea urchin embryo

The most animal part of the ciliated band of sea urchin larvae, the animal plate, is a specialized region in which elongated cells form long and non-beating cilia. To learn how this region is specified, animal halves were isolated from the early cleavage to pregastrulation stages. As is well known, the animal half that is isolated at the eight-cell stage develops into a 'dauerblastula', which forms long and non-beating cilia all around the surface. The region with long cilia, however, became restricted toward the animal pole when separation was delayed. If separated before primary mesenchyme ingression, even a small animal-pole-side fragment formed a normal-sized animal plate. Thus, the prospective animal plate region is gradually restricted by some signal from the vegetal hemisphere, and the specification process terminates before the mesenchyme blastula stage. It was also known that a normal-sized animal plate was formed in micromere-less embryos, indicating that the signal does not depend on micromeres or their descendants. Further, the animal-pole-side fragments were isolated from embryos in which the third cleavage plane was shifted toward the vegetal pole. They formed a normal-sized animal plate, containing more than 75% of the egg volume from the animal pole. This indicates that the egg cytoplasm delivered to veg1-lineage blastomeres plays an important role in the animal plate specification. Interestingly, the an1-less embryo formed long and non-beating cilia at its top region, but thickening did not occur. The cytoplasm near the animal pole might contain some factors necessary for the animal plate to become thick.

Polycomb group gene expression in the sea urchin

Embryonic development in metazoan animals involves a remarkable degree of both genetic and epigenetic regulation. Polycomb group (PcG) genes are essential for initiation and maintenance of epigenetic developmental regulation. This report identifies homologous PcG genes representing the core components of three known Polycomb repressive complexes in the sea urchin Strongylocentrotus purpuratus. Quantitative, temporal, and spatial analyses revealed two significant aspects of their transcript expression during embryonic development. First, almost all showed a localized expression pattern in mesenchyme blastula and gastrula-stage embryos. As the embryo specifies its three germ layers, it may require its PcG gene transcripts to remain in localized stem cell populations, but turn-over in differentiating tissues. Second, their transcript levels gradually decreased during larval pluteus stages. This finding is consistent with progressive cell differentiation in the embryo. Taken together, these results suggest that PcG genes are conserved in S. purpuratus and are actively expressed during early embryogenesis.

Spatio-temporal expression of a Netrin homolog in the sea urchin Hemicentrotus pulcherrimus (HpNetrin) during serotonergic axon extension

A netrin homolog of the sea urchin, Hemicentrotus pulcherrimus (HpNetrin) was characterized in terms of its expression behavior. The predicted amino acid sequence was an ortholog of hemichordate netrin-1. Reverse transcriptase-PCR, immunoblotting, and whole mount immunohistochemistry showed that gene transcription and protein expression occurred from 15-hour post-fertilization (hpf) swimming blastula stage to, at least 53-hpf pluteus stage. HpNetrin was detected on the entire basal surface of the ectoderm in swimming and 16-hpf mesenchyme blastulae. However, by 24-hpf prism stage, immunoreaction on the oral ectoderm decreased, whereas it increased in the aboral ectoderm including near the animal plate ectoderm (area-I). By 48-hpf pluteus stage, the HpNetrin-rich area-I comprised a 40mm wide dorsal midline belt (DMB) that stretched from the dorsal posterior edge of the animal plate to the posterior end of the larval body. Serotonergic cells were first detected in the HpNetrin-moderate area between the anterior DMB and the HpNetrin-poor oral ectoderm near the animal plate in 24-hpf prism larvae. By 48-hpf pluteus stage, these cells extended axons toward the middle-ridge to form a neural plexus of the apical ganglion. At 53-hpf pluteus stage, these axons extended further away from the apical ganglion directly or through the DMB toward the HpNetrin-poor contralateral ectoderm. The protein expression and axon extension pattern were reproduced in embryonic cell-aggregates formed from artificially dissociated 20-hpf gastrulae and resembled small blastula. In Hpnetrin morpholino anti-sense oligonucleotide-injected plutei, serotonergic axon extension was severely inhibited. Thus, HpNetrin functions as a serotonergic axon guidance cue in this basal deuterostome.

Germ line determinants are not localized early in sea urchin development, but do accumulate in the small micromere lineage

Two distinct modes of germ line determination are used throughout the animal kingdom: conditional—an inductive mechanism, and autonomous—an inheritance of maternal factors in early development. This study identifies homologs of germ line determinants in the sea urchin Strongylocentrotus purpuratus to examine its mechanism of germ line determination. A list of conserved germ-line associated genes from diverse organisms was assembled to search the S. purpuratus genome for homologs, and the expression patterns of these genes were examined during embryogenesis by whole mount in situ RNA hybridization and QPCR. Of the 14 genes tested, all transcripts accumulate uniformly during oogenesis and Sp-pumilio, Sp-tudor, Sp-MSY, and Sp-CPEB1 transcripts are also uniformly distributed during embryonic development. Sp-nanos2, Sp-seawi, and Sp-ovo transcripts, however, are enriched in the vegetal plate of the mesenchyme blastula stage and Sp-vasa, Sp-nanos2, Sp-seawi, and Sp-SoxE transcripts are localized in small micromere descendents at the tip of the archenteron during gastrulation and are then enriched in the left coelomic pouch of larvae. The results of this screen suggest that sea urchins conditionally specify their germ line, and support the hypothesis that this mechanism is the basal mode of germ line determination amongst deuterostomes. Furthermore, accumulation of germ line determinants selectively in small micromere descendents supports the hypothesis that these cells contribute to the germ line.