Giovanni Giudice

Restitution of whole larvae from disaggregated cells of sea urchin embryos.

Abstract A method is described which permits the complete isolation of the cells of large numbers of sea urchin embryos, from the blastula to the pluteus stage. Some structural characteristics of the isolated cells are reported. When transferred back to normal sea water, the isolated cells start quickly to reaggregate and form structures which differentiate into quasi-normal larvae. The degree of differentiation attained by these larvae depends upon the stage of the embryo from which the cells were isolated, being higher the younger the embryo.

Synthesis of heat-shock proteins in developing sea urchins.

Heating sea urchin embryos at 31°C greatly reduces the synthesis of the bulk proteins, whereas it highly stimulates the synthesis of some new proteins, the main ones being two closely migrating proteins of about 70,000 daltons. The production of heat-shock proteins is obtained only if the embryos are heated after hatching. Stages which produce heat-shock proteins survive heating, whereas earlier stages, not producing heat-shock proteins, do not survive. Heat-shock proteins are not produced in the presence of actinomycin D.

Synthesis of ribosomal RNA during sea-urchin development

(1) Labelling experiments in vivo followed by sucrose gradient and base analysis of RNA demonstrate that the synthesis of the rRNA extractable from the cytoplasm is at a very low level before gastrulation. At this stage it undergoes a sharp increase. (2) Evidence is provided for the synthesis of a precursor of the cytoplasmic rRNA. (3) The suggestion is made that the increase of rRNA synthesis is due to a preferential synthesis of this type of RNA with respect to other RNA classes.

Gene expression in sea urchin development

SummaryActinomycin D has been administered for various lengths of time and at various concentrations to developing sea urchin embryos. The results obtained support the following suggestions.(1)The shape of respiratory curve up to the mesenchyme blastula stage is not under direct gene control.(2)The RNA needed for gastrulation seems to be made during the period between early and late blastula.(3)The RNA needed for skeleton differentiation is continously made from some time before early gastrula throughout skeleton growth.(4)The process of development of skeleton is extremely sensitive to actinomycin.ZusammenfassungSeeigelkeime wurden während der Frühentwicklung unterschiedlich mit verschiedenen Konzentrationen von Actinomycin D behandelt. — Die Resultate legen folgende Deutungen nahe:1.Der Verlauf der Atmungskurve steht bis zum Stadium der Mesenchymblastula nicht unter genetischer Kontrolle.2.Die zur Gastrulation benötigte RNS dürfte in der Zeit zwischen früher und später Blastula gebildet werden.3.Die zur Differenzierung des Skeletts benötigte RNS wird im Verlauf des Skelettwachstums gebildet; die Synthese beginnt kurz vor der Gastrulation.4.Der Prozeß der Skelettentwicklung ist gegen Actinomycin extrem empfindlich.

A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed.

In the present study, double immunofluorescence and immunoblot analysis have been used to show that centrosomes, isolated from Paracentrotus lividus sea urchin embryos at the first mitotic metaphase, contain the constitutive chaperone, heat-shock protein (HSP) 70. More specifically, we demonstrate that centrosomes contain only the HSP70-d isoform, which is one of the four isoforms identified in P. lividus. We also provide evidence that p34(cell division control kinase-2) and t complex polypeptide-1 (TCP-1) alpha, a subunit of the TCP-1 complex, are localized on the centrosomes. Furthermore, inhibition of TCP-1 in vivo, via microinjecting an anti-(TCP-1 alpha) antibody into P. lividus eggs before fertilization, either impaired mitosis or induced severe malformations in more than 50% of embryos. In addition, we have isolated the whole mitotic apparatus and shown that HSP70 localizes on the fibres of spindles and asters, and binds them in an ATP-dependent manner. These observations suggest that HSP70 has a chaperone role in assisting the TCP-1 complex in tubulin folding, when localized on centrosomes, and during the assembling and disassembling of the mitotic apparatus, when localized on the fibres of spindles and asters.

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. Intra‐ embryonic 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.

Synthesis of ribosomal RNA in disaggregated cells of sea urchin embryos

Abstract The specific activity of the α-phosphate of the acid-soluble nucleotide pool has been found to remain constant for several hours after the dissociation into cells of embryos previously exposed to inorganic 32 P. The rate of incorporation of 32 P into 28-S rRNA of dissociated cells deriving from prelabeled embryos has been used as a measure of the rate of rRNA synthesis. The results strongly suggest that neither intercellular contact nor cell division, at least from the stage of hatching on, are needed for the activation of rRNA synthesis, characteristic of the normal development, to take place.

Synthesis of ribosomal RNA during sea urchin development: III. Evidence for an activation of transcription

Abstract Embryos of Paracentrotus lividus have been preloaded with 32P1 until the early blastula stage. The amount of the total acid-soluble nucleotide pool, as well as the specific activity of the α-phosphates of such nucleotides, were found to remain practically constant until at least the prism stage. The specific activity attained at different stages by the 28-S rRNA was calculated and referred to the total DNA content of the embryos. An activation of rRNA transcription following the swimming blastula stage has been demonstrated.

Studies on sea urchin oocytes II. Synthesis of RNA during oogenesis

Abstract Isolated oocytes of the sea urchin Paracentrotus lividus actively incorporate 3H-uridine into RNA. Labeled RNA was analysed by sucrose gradient and acrylamide gel electrophoresis following cell fractionation. Much of the radioactivity is incorporated at the nucleolar level in the form of rRNA precursors. The kinetics of maturation of these latter suggests that this occurs at a slower rate than during embryogenesis. Other non-nucleolar RNA classes are also actively labelled and retained in the nucleus for many hours. These results are confirmed by an autoradiographic investigation.

Studies on sea urchin oocytes: I. Purification and cell fractionation☆

Abstract A procedure is presented which permits a satisfactory purification in bulk of oocytes of Paracentrotus lividus at various stages in the growth period. Methods which allow a quantitative recovery of partially purified germinal vesicles or nucleoli are also reported.