Development of expressed sequence tag-derived microsatellite markers for the sea urchin Hemicentrotus pulcherrimus

The first set of 15 expressed sequence tag-simple sequence repeat markers was developed in sea urchin Hemicentrotus pulcherrimus. Number of alleles per locus ranged from two to 17. The observed and expected heterozygosities ranged from 0.022 to 0.911 and from 0.022 to 0.916, respectively. These informative marker loci will be useful for the assessment of genetic variation and population structure of this species.     

青岛近岸海域马粪海胆摄食的实验生态学研究

对采自青岛近岸海域的马粪海胆从其对各种饵料的选择和摄食吸收、温度与自身湿重对其摄食率的影响以及摄食活动特征等方面进行实验生态学研究。结果表明 ,马粪海胆在多种海藻并存的情况下 ,对海带及裙带菜等褐藻具明显的选择性 ,对石花菜及孔石莼等藻类的喜好性较差 ,但在食物匮乏条件下 ,马粪海胆的食谱则变得相当广泛。马粪海胆对裙带菜及海带的摄食率均明显高于孔石莼 ,但其对孔石莼的饵料吸收率则高于海带及裙带菜。马粪海胆的摄食受温度条件及自身湿重的影响 ,温度偏离适宜范围对摄食有抑制作用 ,日摄食率与湿重呈显著的指数下降关系 ,摄食率与湿重、温度因子可建立具较高可靠性的指数回归模型。马粪海胆对食物的口面吸附与对其摄食密切相关 ,其摄食活动与湿重呈显著的指数下降关系 ,而与排便活动呈显著的正相关 ,随湿重增加排便活动虽呈下降趋势 ,但未达显著水平。自然光照条件下马粪海胆的夜间摄食强度通常高于日间 ,但在全遮盖的无光照条件下这种昼夜摄食差异则变得不显著 ;对摄食活动的连续观测表明马粪海胆的摄食活动受光强变化的影响 ,其摄食活动的高峰出现在光强减弱的早间及傍晚 ,而日间的强光照环境会抑制其摄食活动     

A laboratory study of the correlation between texture and the speed of locomotion by the sea urchin Hemicentrotus pulcherrimus

This laboratory study examined the relationship between substrate texture and movement speed of the sea urchin Hemicentrotus pulcherrimus. We assessed the movement speed of 14 sea urchins placed on either acrylic, or three types of waterproof sandpaper, on the bottom of a water tank. Images were taken at regular intervals and analyzed to determine the speed of each sea urchin. Light intensity was stronger at one end of the tank. Our analysis showed sea urchins moved away from light, at a speed that was negatively correlated with the roughness of the substrate, with slower movement on rougher surfaces. This result has implications for the design of equipment for capturing sea urchins in areas where their explosive population growth presents a threat to algal growth and reef environments.     

Studies on heat shock proteins in sea urchin development

Work on stress proteins in sea urchin embryos carried out over the last 20 years is reviewed and the following major results are described. Entire sea urchin embryos, if subjected to a rise in temperature at any postblastular stage undergo a wave of heat shock protein (hsp) synthesis and survive. If subjected to the same rise between fertilization and blastula formation, they are not yet able to synthesize hsp and die. Four clones coding for the major hsp, hsp70, have been isolated and sequenced; evidence for the existence of a heat shock factor has been provided, and a mechanism for the developmental regulation of hsp synthesis discussed. Intraembryonic and intracellular hsp location has been described; and a mechanism for achievement of thermotolerance proposed. A chaperonine role for a constitutive mitochondrial hsp56 has been suggested, as well as a role for the constitutive hsp70 in cell division. Heat shock, if preceded by 12-O-tetradecanoylphorbol-12-acetate (TPA) treatment causes apoptosis.     

Changes in cAMP-dependent protein kinase activity in the 10,000g sediment of sea urchin embryos during early development

cAMP-dependent protein kinase was found in the sediment obtained by centrifuging a homogenate of sea urchin embryos at 10,000g for 20 min, and was solubilized with 1% Triton X-100. This enzyme was eluted at 0.16 M NaCl in a linear concentration gradient on a DEAE-cellulose column, at which cAMP-dependent protein kinase found in the supernatant was also eluted. The enzyme activity was enhanced about 1.5-fold in the presence of 1 μM cAMP, and increased somewhat by adding cGMP or cIMP. The activation by cAMP of protein kinase in the sedimentable fraction was lower than in the supernatant fraction. The properties of the enzyme found in the 10,000g sediment and in the supernatant differ somewhat. The activity of the cAMP-dependent protein kinase in the 10,000g sediment was high in the embryos at the blastula, the swimming blastula, and the mesenchyme blastula stages. On the other hand, the activity was undetectable in unfertilized eggs and in embryos at the morula, the gastrula, and the pluteus stages.     

A developmentally regulated α2,8-polysialyltransferase in embryos of the sea urchin Lytechinus pictus

The recent chemical identification of polysialylated glycoproteins in the jelly coat and on the cell surface of the sea urchin egg raises important questions about their biosynthesis and possible function. Using CMP-[¹⁴C]-Neu5Ac as substrate and cell free preparations from eggs and embryos of the sea urchin Lytechinus pictus , we have identified a membrane associated CMP-Neu5Ac:poly-α2,8 sialosyl sialyltransferase (polyST) that transferred Neu5Ac from CMP-Neu5Ac to an endogenous acceptor membrane protein of approximately 38kDa. An average of five to six [¹⁴C]-Neu5Ac residues were transferred to the glycan moiety of this protein. The membrane-associated polyST also catalyzed the polysialylation of several exogenous mammalian ganglioside acceptors, including G_D3. Given that no structurally similar naturally occurring polysialylated gangliosides have been described, nor were observed in the present study, we conclude that a single polyST activity catalyzes sialylation of the endogenous acceptor protein and the gangliosides. Using an excess of G_D3 as an exogenous acceptor, it was established that the expression of the polyST in L. pictus embryos increased rapidly at the mesenchyme blastula stage and reached a maximum at the gastrula stage. The finding that this polyST in the sea urchin embryo is developmentally regulated raises the possibility that it may play a role in the changing cell and tissue interactions that occur during gastrulation and the early stages of spicule formation.     

Phosphorylation-dependent regulation of skeletogenesis in sea urchin micromere-derived cells and embryos

Sea urchin embryo micromeres when isolated and cultured in vitro differentiate to produce spicules. Although several authors have used this model, almost nothing is known about the signaling pathways responsible for initiating skeletogenesis. In order to investigate the potential involvement of phosphorylation events in spiculogenesis, the effect of inhibitors of protein kinases and phosphatases on skeleton formation was studied. Results obtained using both cultured micromeres and embryos revealed that protein tyrosine kinase and phosphatase inhibitors blocked skeleton formation, but not serine/threonine phosphatase inhibitors. The inhibitors showed a dose-dependent effect and when removed from micromere or embryo culture, spicule formation resumed. Inhibition of tyrosine phosphatases resulted in an increase in the tyrosine phosphorylation level of two major proteins and a modest decrease in the expression of the mRNA coding for type I fibrillar collagen. These findings strongly suggest that tyrosine phosphorylation and dephosphorylation is required for micromere differentiation and for normal skeletogenesis during sea urchin embryo development.     

Changes in the activities of protein phosphatase type 1 and type 2A in sea urchin embryos during early development

In the eggs and embryos of sea urchins, the activity of protein phosphatase type 2A (PP2A) increased during the developmental period between fertilization and the morula stage, decreased after the prehatching blastula stage and increased again after hatching. The PP2A activity changed keeping pace with alteration to the activities of cAMP-dependent protein kinase (A kinase), Ca2+/calmodulin-dependent protein kinase (CaM kinase) and casein kinase. Probably, PP2A contributes to the quick turning off of cellular signals because of protein phosphorylation. The activity of protein phosphatase type 1 (PP1) was not detectable up to the morula stage and appreciably increased thereafter. In the isolated nucleus fraction, specific activities of PP1 and PP2A were higher than in whole embryos at all stages in early development. Exponential increase in the number of nuclei because of egg cleavage probably makes PP1 activity detectable in whole embryos after the morula stage. In isolated nuclei, the activities of PP1 and PP2A appreciably decreased after hatching, whereas the activities of A kinase, Ca2+/phospholipid-dependent protein kinase (C kinase) and CaM kinase, as well as casein kinase, became higher. In nuclei, cellular signals caused by protein phosphorylation after hatching do not seem to be turned off by these protein kinases so quickly as before hatching. The PP1 and PP2A in nuclei also seem to contribute to the elimination of signal noise.     

Complete regulation of development throughout metamorphosis of sea urchin embryos devoid of macromeres

The developmental potential of the animal cap (consisting of eight mesomeres) recombined with micromeres or of micromere progeny was examined in sea urchin embryos. The embryos derived from the animal cap recombined with a quartet of micromeres or their descendants developed into four-armed plutei. After feeding, the larvae developed into eight-armed plutei. The left-right polarity of the larvae, recognized by the location of the echinus rudiment, was essentially normal, regardless of the orientation of animal-vegetal polarity in micromeres combining with the animal cap. The larvae had sufficient potential to metamorphose into complete juvenile sea urchins with five-fold radial symmetry. Cell lineage tracing experiments showed that: (i) macromere progeny were not required for formation of the typical pattern of primary mesenchyme cells derived exclusively from large micromeres; (ii) the progeny of large micromeres did not contribute to cells in the endodermal gut with three compartments of normal function; (iii) the presumptive ectoderm had the potential to differentiate into endodermal gut and mesodermal secondary mesenchyme cells, from which pigment cells likely differentiated; and (iv) behavior of the progeny of small micromeres was the same as that in normal embryos through the gastrula stage. These results indicate that the mesomeres respecify their fate under the inductive influence of micromeres so perfectly that complete juvenile sea urchins are produced.     

cAMP-dependent protein kinase in sea urchin embryos

cAMP-dependent protein kinase in the supernatant fraction of the homogenate of sea urchin eggs and embryos obtained by centrifugation at 105,000g was investigated in the present study. In the previous report, the dissociation constant between cAMP-binding proteins and cAMP changed during the development. This suggests that the nature of cAMP-dependent protein kinase, which has been well established to be the major cAMP receptor, changes during the development. In the present study, four protein kinases were separated through DEAE-cellulose column from the supernatant of unfertilized egg homogenate. One of them was cAMP-dependent protein kinase. The others were cAMP-independent ones. One among them was phosvitin kinase, and the others were not identified at present. The activity of cAMP-dependent protein kinase gradually increased during a period from fertilization to the swimming blastula stage. During this period, cleavages occurred at a high rate, and the rate decreased after hatching out. Thus, it is supposed that cAMP-dependent protein kinase in the supernatant may take a part in the mechanism of cleavage. The activity, however, became very low at the mesenchyme blastula, the gastrula, and the pluteus stages. cAMP-binding capacity was observed in the sedimentable fraction and the supernatant fraction, respectively, obtained by 105,000g centrifugation at all stages examined. If the structure-bound cAMP-binding protein is also cAMP-dependent protein kinase, it may play different roles in the mechanism of development.     

Inhibition of sea urchin fertilization by jaspisin, a specific inhibitor of matrix metalloendoproteinase

Jaspisin, originally isolated from a marine sponge as an inhibitor of the hatching of the sea urchin (Hemicentrotus pulcherrimus) embryo, causes inhibition of sea urchin fertilization. Electron microscopic examination revealed that the acrosome reaction was induced in jaspisin-treated sperm when they were incubated with an intact egg. The acrosome-reacted sperm bound to the vitelline layer by the acrosomal material surrounding the acrosomal process. However, fusion of the acrosomal process and the egg plasma membrane failed to take place. Membrane potential changes were monitored using eggs preloaded with a membrane potential-sensitive fluorochrome, di-8-ANEPPS. Depolarization of the membrane potential, normally observed in the fertilized egg was not observed in the egg inseminated in the presence of jaspisin, indicating the absence of electrical continuity between the jaspisin-treated egg and sperm. Jaspisin inhibited the activities of matrix metallo-endoproteinase members but not of other types of proteinases. These results provide strong, albeit indirect, evidence that a matrix metallo-endoproteinase(s) is involved in the process of gamete fusion during sea urchin fertilization.     

Initial analysis of immunochemical cell surface properties, location and formation of the serotonergic apical ganglion in sea urchin embryos

In the present study it was found that serotonergic apical ganglion (SAG)-forming cells in plutei of the sea urchin, Hemicentrotus pulcherrimus, possessed a characteristic pear shape with broad apical sides and a pointed basal side in the acron epithelium. The basal side extended axons through the space between the epithelium and the basal lamina toward the midline of the embryo that aligned parallel to the embryonic anteroposterior axis. Serotonergic apical ganglion-forming cells had epithelial cell surface-specific proteins on their entire surface. The SAG in 4-arm plutei was composed of a 4-cell trunk region that aligned at right angles to the embryonic anteroposterior axis, and forked into two branches of one to two cells at both ends. Two branches extended toward the oral and the other two toward the aboral region, respectively. Double-stained immunohistochemistry using antiserotonin antibodies and oral ectoderm-specific anti-Ecto V monoclonal antibody or aboral ectoderm-specific anti-Ars antibodies indicated that SAG was in the aboral ectoderm region. Serotonergic apical ganglion cells were first detected in late gastrulae and increased in number rapidly between 36 and 48 h after fertilization, and then slowly afterwards. A 5-bromo-2-deoxyuridine incorporation study indicated that none of the increased SAG cells were in the S phase during the aforementioned period, suggesting that SAG cells do not proliferate by cell division, but acquire the property in particular cells by transdifferentiation using a mechanism that has yet to be elucidated.     

马粪海胆对环境变化的耐受性与选择性研究

对采自青岛近岸的马粪海胆进行温度、盐度、光强与底质等因子的耐受及选择实验。结果表明,青岛近岸马粪海胆的适温范围约在8-22℃,对温度的选择受驯养水温的影响;属窄盐性种类,适盐范围约在30-35;喜好弱光环境,饥饿状态的选择光强（8-25lx）较非饥饿状态（10-35lx）低;对粗沙砾底质具明显的正选择,而对细沙性底质呈明显的负选择。     

Functional organization of DNA elements regulating SM30α, a spicule matrix gene of sea urchin embryos

The SM30a gene encodes a protein in the embryonic endoskeleton of the sea urchin Strongylocentrotus purpuratus, and is specifically expressed in the skeletogenic primary mesenchyme cell lineage. To clarify the mechanism for the differentiation of this cell lineage, which proceeds rather autonomously in the embryo, regulation of the SM30alpha gene was investigated previously and it was shown that the distal DNA region upstream of this gene from - 1.6 to - 1.0 kb contained numerous negative regulatory elements that suppressed the ectopic expression of the gene in the gut. Here we study the influence of the proximal region from - 303 to + 104 bp. Analysis of the expression of reporter constructs indicated that a strong positive enhancer element existed in the region from -142 to -105bp. This element worked both in forward and reverse orientations and additively when placed tandemly upstream to the reporter gene. In addition, other weaker positive and negative regulatory sites were also detected throughout the proximal region. Electrophoretic gel mobility shift analyses showed that multiple nuclear proteins were bound to the putative strong enhancer region. One of the proteins binding to this region was present in ear y blastulae, a time when the SM30 gene was still silent, but it was not in prism embryos actively expressing the gene. The binding region for this blastula-specific protein was narrowed down to the region from - 132 to -122 bp, which included the consensus binding site for the mammalian proto-oncogene product, Ets. Two possible SpGCF1 binding sites were identified in the vicinity of the enhancer region. This information was used to make a comparison of the general regulatory architecture of genes that contribute to the formation of the skeletal spicule.     

Commitment to vegetalized development in sea urchin embryos

Summary Strongylocentrotus purpuratus embryos were reared in 0.025 M LiCl, which causes commitment to vegetalized development within 5 h after treatment begun at fertilization. Treated and control embryos were labelled with³⁵S-methionine for 3 h intervals from 2–14 h, solubilized, and subjected to 2-dimensional polyacrylamide gel electrophoresis. Comparison of autoradiographs of the gels, in which over 400 proteins can be detected, indicate that while LiCl treatment causes a short delay in the initiation or cessation of synthesis of a few proteins, no qualitative or major quantitative differences can be detected between control and treated embryos. Normal gastrulae and vegetalized exogastrulae labelled 38 h after fertilization have several differences in patterns of protein synthesis. We conclude that the early determinative events involved in vegetalization are not reflected in detectable differences in the pattern of protein synthesis.     

Zymogen activation and characterization of a major gelatin-cleavage activity localized to the sea urchin extraembryonic matrix

The hyaline layer (HL) is an apically located extracellular matrix (ECM) which surrounds the sea urchin embryo from the time of fertilization until metamorphosis occurs. While gelatin-cleavage activities were absent from freshly prepared hyaline layers, a dynamic pattern of activities developed in layers incubated at 15 or 37 degrees C in Millipore-filtered sea water (MFSW). Cleavage activities at 90, 55, 41, and 32 kDa were evident following incubation at either temperature. The activation pathway leading to the appearance of these species was examined to determine the minimum salt conditions required for processing and to establish precursor-product relationships. In both qualitative and quantitative assays, the purified 55 kDa gelatinase activity was inhibited by 1,10-phenanthroline (a zinc-specific chelator) and ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA). Calcium reconstituted the activity of the EGTA-inhibited enzyme with an apparent dissociation constant (calcium) of 1.2 mM. Developmental substrate gel analysis was performed using various stage embryos. The 55 and 32 kDa species comigrated with gelatin-cleavage activities present in sea urchin embryos. Collectively, the results reported here document a zymogen activation pathway which generates a 55 kDa, gelatin-cleaving activity within the extraembryonic HL. This species displayed characteristics of the matrix metalloproteinase class of ECM modifying enzymes.     

Change in phosvitin kinase activity during early development of the sea urchin

Protein kinase, which phosphorylated phosvitin at the expense of ATP but did not phosphorylate casein, protamine, and histone mixture, was obtained by DEAE-cellulose column chromatography of the extract from the embryos of the sea urchin, Strongylocentrotus intermedius. This enzyme, partially purified by DEAE-cellulose column, reversibly catalyzed the reaction of phosvitin phosphorylation. This indicates that the sea urchin embryos contain phosvitin kinase. Phosvitin kinase in sea urchin embryos is somewhat different from that found in the other types of cells, which are able to phosphorylate casein as well as phosvitin. In unfertilized eggs, the activity of this enzyme was found only in the supernatant fraction obtained by centrifuging the homogenate at 10,000g for 20 min. The activity in the embryos at the swimming and the mesenchyme blastula stage was higher than in unfertilized eggs, and was localized in the sedimentable fraction obtained by centrifuging the homogenate of the embryos at 10,000g for 20 min. The highest activity of phosvitin kinase was observed in the embryos at the mesenchyme blastula stage, and the enzyme activity became quite low at the late gastrula stage. The activity and the intracellular distribution of phosvitin kinase changed during the development. The enzyme in this sedimentable fraction was not solubilized with 1% Triton X-100 but was extracted by 1 M NaCl.     

A new G-stretch-DNA-binding protein, Unichrom, displays cell-cycle-dependent expression in sea urchin embryos

We report the identification and characterization of Unichrom, a gene encoding a new G-stretch-DNA-binding protein in the sea urchin embryo. The derived amino acid sequence of Unichrom contains plant homeodomain (PHD) finger and high mobility group (HMG) motifs as well as motifs required for cell-cycle-dependent degradation. The expression of a Unichrom-green fluorescent protein (GFP) fusion protein in sea urchin embryonic cells indicates that Unichrom protein accumulates in nuclei during interphase and disperses into the cytoplasm at mitosis. Overexpression of dominant negative Unichrom, which contains the DNA binding domain lacking the motif for cell-cycle-dependent degradation, causes impairment of chromosome segregation. These results suggest that Unichrom binds to genome DNA at G-stretch and that degradation of Unichrom is required for segregation of chromosomes.