Autonomous expression of tissue-specific genes in dissociated sea urchin embryos

Development ◽  
1989 ◽  
Vol 107 (2) ◽  
pp. 299-307 ◽  
Author(s):  
L. Stephens ◽  
T. Kitajima ◽  
F. Wilt
Keyword(s):  
Early Development ◽  
Sea Urchin ◽  
Single Cells ◽  
Cell Types ◽  
Cell Interactions ◽  
Cell Stage ◽  
Tissue Specific ◽  
Specific Transcript ◽  
Sea Urchin Embryos ◽  
Early Stages

The effects of disrupting cell interactions in early development were investigated by examining the accumulation of a primary mesenchyme specific transcript (SM50) and an aboral ectoderm-specific transcript (Spec 1) in cultures of sea urchin embryos that were dissociated at early stages and then cultured in CFSW. The expression of both SM50 and Spec 1 is temporally correct and remains restricted to the appropriate cell types, even if the embryo is dissociated as early as the 2-cell stage and maintained as a suspension of single cells. This result is consistent with the idea that the specificity of expression of these two genes, each characteristic of different lineages, is strongly regulated by information in the egg. Average SM50 expression is half that of intact embryos, but Spec 1 expression is very low, only 10–20% of intact controls, suggesting some differences in the response of the two genes to lack of close cell interactions.

scholarly journals Proteolysis of the major yolk glycoproteins is regulated by acidification of the yolk platelets in sea urchin embryos

1992 ◽  
Vol 117 (6) ◽  
pp. 1211-1221 ◽  
Author(s):  
SK Mallya ◽  
JS Partin ◽  
MC Valdizan ◽  
WJ Lennarz
Keyword(s):  
Early Development ◽  
Sea Urchin ◽  
Ph Value ◽  
Total Protein Content ◽  
Developmentally Regulated ◽  
Cell Stage ◽  
Ph Optimum ◽  
Sea Urchin Embryos

The precise function of the yolk platelets of sea urchin embryos during early development is unknown. We have shown previously that the chemical composition of the yolk platelets remains unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and total protein content after fertilization and early development. However, the platelet is not entirely static because the major 160-kD yolk glycoprotein YP-160 undergoes limited, step-wise proteolytic cleavage during early development. Based on previous studies by us and others, it has been postulated that yolk platelets become acidified during development, leading to the activation of a cathepsin B-like yolk proteinase that is believed to be responsible for the degradation of the major yolk glycoprotein. To investigate this possibility, we studied the effect of addition of chloroquine, which prevents acidification of lysosomes. Consistent with the postulated requirement for acidification, it was found that chloroquine blocked YP-160 breakdown but had no effect on embryonic development. To directly test the possibility that acidification of the yolk platelets over the course of development temporally correlated with YP-160 proteolysis, we added 3-(2,4-dinitroanilo)-3-amino-N-methyldipropylamine (DAMP) to eggs or embryos. This compound localizes to acidic organelles and can be detected in these organelles by EM. The results of these studies revealed that yolk platelets did, in fact, become transiently acidified during development. This acidification occurred at the same time as yolk protein proteolysis, i.e., at 6 h after fertilization (64-cell stage) in Strongylocentrotus purpuratus and at 48 h after fertilization (late gastrula) in L. pictus. Furthermore, the pH value at the point of maximal acidification of the yolk platelets in vivo was equal to the pH optimum of the enzyme measured in vitro, indicating that this acidification is sufficient to activate the enzyme. For both S. purpuratus and Lytechinus pictus, the observed decrease in the pH was approximately 0.8 U, from 7.0 to 6.2. The trypsin inhibitor benzamidine was found to inhibit the yolk proteinase in vivo. By virtue of the fact that this inhibitor was reversible we established that the activity of the yolk proteinase is developmentally regulated even though the enzyme is present throughout the course of development. These findings indicate that acidification of yolk platelets is a developmentally regulated process that is a prerequisite to initiation of the catabolism of the major yolk glycoprotein.

A fate map of the vegetal plate of the sea urchin (Lytechinus variegatus) mesenchyme blastula

Development ◽  
1996 ◽  
Vol 122 (1) ◽  
pp. 253-263 ◽  
Author(s):  
S.W. Ruffins ◽  
C.A. Ettensohn
Keyword(s):  
Sea Urchin ◽  
Single Cells ◽  
Cell Types ◽  
Lineage Tracing ◽  
Lytechinus Variegatus ◽  
Fate Map ◽  
Blastula Stage ◽  
Cell Stage ◽  
Mesodermal Cell ◽  
Single Blastomeres

Previous lineage tracing experiments have shown that the vegetal blastomers of cleavage stage embryos give rise to all the mesoderm and endoderm of the sea urchin larva. In these studies, vegetal blastomers were labeled no later than the sixth cleavage division (60-64 cell stage). In an earlier study we showed that single cells in the vegetal plate of the blastula stage Lytechinus variegatus embryo could be labeled in situ with the fluorescent, lipophilic dye, DiI(C18), and that cells labeled in the central region of the vegetal plate of the mesenchyme blastula primarily gave rise to homogeneous clones consisting of a single secondary mesenchyme cell (SMC) type (Ruffins and Ettensohn (1993) Dev. Biol. 160, 285–288). Our clonal labeling showed that a detailed fate map could be generated using the DiI(C18) labeling technique. Such a fate map could provide information about the spatial relationships between the precursors of specific mesodermal and endodermal cell types and information concerning the movements of these cells during gastrulation and later embryogenesis. We have used this method to construct the first detailed fate map of the vegetal plate of the sea urchin embryo. Ours is a latitudinal map; mapping from the plate center, where the mesodermal precursors reside, through the region which contains the endodermal precursors and across the ectodermal boundary. We found that the precursors of certain SMC types are segregated in the mesenchyme blastula stage vegetal plate and that prospective germ layers reside within specific boundaries. To determine whether the vegetal plate is radially symmetrical with respect to mesodermal cell fates, single blastomeres of four cell stage embryos were injected with lysyl-rhodamine dextran (LRD). The resulting ectodermal labeling patterns were classified and correlated with the SMC types labeled. This analysis indicates that the dorsal and ventral blastomers do not contribute equally to SMC derivatives in L. variegatus.

scholarly journals SYNTHESIS AND STORAGE OF MICROTUBULE PROTEINS BY SEA URCHIN EMBRYOS

1971 ◽  
Vol 50 (2) ◽  
pp. 516-528 ◽  
Author(s):  
Rudolf A. Raff ◽  
Gerald Greenhouse ◽  
Kenneth W. Gross ◽  
Paul R. Gross
Keyword(s):  
Amino Acids ◽  
Sea Urchin ◽  
Messenger Rna ◽  
Binding Activity ◽  
Total Soluble Protein ◽  
Cell Stage ◽  
Lytechinus Pictus ◽  
Sea Urchin Embryos ◽  
Tritiated Amino Acids ◽  
Vinblastine Sulfate

Studies employing colchicine binding, precipitation with vinblastine sulfate, and acrylamide gel electrophoresis confirm earlier proposals that Arbacia punctulata and Lytechinus pictus eggs and embryos contain a store of microtubule proteins. Treatment of 150,000 g supernatants from sea urchin homogenates with vinblastine sulfate precipitates about 5% of the total soluble protein, and 75% of the colchicine-binding activity. Electrophoretic examination of the precipitate reveals two very prominent bands. These have migration rates identical to those of the A and B microtubule proteins of cilia. These proteins can be made radioactive at the 16 cell stage and at hatching by pulse labeling with tritiated amino acids. By labeling for 1 hr with leucine-3H in early cleavage, then culturing embryos in the presence of unlabeled leucine, removal of newly synthesized microtubule proteins from the soluble pool can be demonstrated. Incorporation of labeled amino acids into microtubule proteins is not affected by culturing embryos continuously in 20 µg/ml of actinomycin D. Microtubule proteins appear, therefore, to be synthesized on "maternal" messenger RNA. This provides the first protein encoded by stored or "masked" mRNA in sea urchin embryos to be identified.

Synthesis of nuclear and cytoplasmic proteins in the early development of fishes and echinoderms

Development ◽  
1971 ◽  
Vol 26 (3) ◽  
pp. 611-622
Author(s):  
Maya R. Krigsgaber ◽  
Alla A. Kostomarova ◽  
Tamara A. Terekhova ◽  
Tatiana A. Burakova
Keyword(s):  
Amino Acids ◽  
Early Development ◽  
Sea Urchin ◽  
Nuclear Volume ◽  
Sea Urchin Embryos ◽  
Cytoplasmic Proteins ◽  
Specific Activities ◽  
Strongylocentrotus Nudus ◽  
Silver Grains ◽  
Misgurnus Fossilis

Synthesis of nuclear and cytoplasmic proteins was studied biochemically and autoradiographically in early loach (Misgurnus fossilis) and sea-urchin (Strongylocentrotus nudus) embryos. After incubation with [14C]amino acids for 5–120 min the ratio of the specific activities of nuclear, mitochondrial and 12000 g supernatant proteins was shown to be equal approximately to 6:1:2 in loach embryos and to 8:4:3 in sea-urchin embryos independently of the duration of labelling. After incubation with [3H]amino acids the number of silver grains per unit section was on the average 2·4 times higher for nuclei than it was for cytoplasm at mid-blastula and mid-gastrula stages. At the mid-gastrula the vegeto-animal gradient of protein synthesis was found. A higher level of the synthesis of nuclear proteins as compared with that of cytoplasmic proteins appears to be related to an increase in the nuclear volume and the nucleo-cytoplasmic ratio during the early development of the loach and sea-urchin embryos.

Properties of the three cell types in sixteen-cell sea urchin embryos: Aggregation and microtuble protein synthesis

1972 ◽  
Vol 27 (2) ◽  
pp. 150-164 ◽  
Author(s):  
Richard O. Hynes ◽  
Rudolf A. Raff ◽  
Paul R. Gross
Keyword(s):  
Protein Synthesis ◽  
Sea Urchin ◽  
Cell Types ◽  
Sea Urchin Embryos

scholarly journals ADP-ribosyltransferase in isolated nuclei from sea-urchin embryos

1985 ◽  
Vol 225 (2) ◽  
pp. 429-434 ◽  
Author(s):  
A Isoai ◽  
I Yasumasu
Keyword(s):  
Sea Urchin ◽  
Insoluble Fraction ◽  
Cell Stage ◽  
Isolated Nuclei ◽  
Sea Urchin Embryos ◽  
Snake Venom Phosphodiesterase ◽  
Km Value ◽  
Insoluble Product ◽  
Adp Ribosyltransferase ◽  
Hydrolysis Of

The activity of ADP-ribosyltransferase in nuclei isolated from sea-urchin embryos was estimated by the incorporation of [adenosine-14C]NAD+ into the acid-insoluble fraction. Hydrolysis of this acid-insoluble product by snake venom phosphodiesterase yielded radioactive 5′-AMP and phosphoribosyl-AMP. The incorporation of [14C]-NAD+ was inhibited by 3-aminobenzamide and nicotinamide, potent inhibitors of ADP-ribosyltransferase. [14C]NAD+ incorporation into the acid-insoluble fraction results from the reaction of ADP-ribosyltransferase. The optimum pH for the enzyme in isolated nuclei was 7.5. The enzyme, in 50 mM-Tris/HCl buffer, pH 7.5, containing 0.5 mM-NAD+ and 0.5 mM-dithiothreitol, exhibited the highest activity at 18 degrees C in the presence of 14 mM-MgCl2. The apparent Km value for NAD+ was 25 microM. The activity of the enzyme was measured in nuclei isolated from the embryos at several stages during early development. The activity was maximum at the 16-32-cell stage and then decreased to a minimum at the mesenchyme blastula stage. Thereafter its activity slightly increased at the onset of gastrulation and decreased again at the prism stage.

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