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The cholinergic properties of the gastrulating chick embryo
Cell Biology
172
THE CHOLINERGIC GASTRULATING
P373
CHICK
PROPERTIES ENGRYO
OF
THE
Tiit Laasherg, Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 200026, Estonia The gastrulating chick embryo has the main cholinergic characteristics whose acetylcholinpresence is indicated by esterase (AChE, EC 3.1.1.7) and choline acetyltransferase (CAT, EC 2.3.1.6) activand by the existence of functionally ity, active muscarinic receptors which are phosphatidylinositol turncoupled with over. A sharp increase in AChE activity occured in the gastrulating embryo. The highest activity was associated with hypoblast cells. By sucrose density gradient centrifugation three molecular forms of AChE with sedimentation coefficients 4.7S, 6.8s and 10.9s were determined. During the gastrulation there was no remarkable change in the activity of CAT. concentration The intracellular of acetylcholine (ACh) in gastrulating chick
embryo cells was about 10-6-10-5 M. ACh, via muscarinic receptors increased the from level of free calcium (tCa2+li) the basal level of 120 nM to the 200 nM (measand the level of InsP3. ured with indo-l), The establishment of dose-response curve by the measurement of [Ca2+Ii mobilization in dissociated embryo stages 3 and 4-5 by Hamburger (1951) gave the ED50 value for * 10-e M.
cells at and Hamilton ACh 4cLO.5)
STEADY IONIC CURRENTS AROUND THE UNFERTILISED EGG OF THE SEA URCHIN, HOLOPNEUSTES PURPURESCENS.
P375
Jane M. Chrystal, Robyn L. Overall and Valerie B. Morris. School of Biological Sciences, University of Sydney, Australia, 2006. A substantial part of our understanding of early animal
development
comes
from
studies
of
sea urchin
eggs and embryos. To date, there have been no reports of sea urchin eggs having steady ionic currento those reported for plant and other ts. similar animal embryos, where the currents have been shown to have developmental significance (Jaffe, 1981). The lack of such reports is probably because the sea urchins studied in North America and Europe have small eggs in the range, 40-lOOurn, that would make the measurement of steady ionic currents difficult. An Australian sea urchin, Holopneustes purpurescens, is a direct developing sea urchin with eggs approximately 400um in diameter. Their especially large size has opened the way for a study of steady ionic currents in sea urchins. Steady ionic currents were measured around these
eggs using Nuccitelii,
the vibrating 1974).
Steady
probe apparatus ionic
currents
(Jaffe
and
up to
2.4uA/cm were measured around the unfertilised eggs of Ii. purpurescens. The pattern of currents tend to be aroused as areas of inward or outward currents. ?he patterns are not mosaics. The data fits a model in which current enters half the egg The current patterns and leaves the other half. appear not to be associated with the area of grey pigmentation of the eggs or with the orientation of The ability to the epg with respect to gravity. measure steady ionic currents is limited by a low signal to noise ratio caused by the high conductivity of seawater. Ours is the first report of the measurement steady ionic currents around a marine
animal.
international
Reports,
Vol. 14, Abstracts
Supplement
1990
CHANGESOFMET~IONEIN CONTRN!l! IN SEAURCHINEMBRYOS B. De Petrocellis, P. De C. Capasso, Prisco, E. Gargiulo, E. Parisi, R. Scudiero. CM Institute of Protein Biochemistry and Enzymology, Zoological Station, 80121 Napoli, Italy
P374
We have studied the changes of the metallothionein (MT) content during development of the sea urchin egg. An extract prepared from unfertilized eggs was fractionated by gel filtration chromatography on a Sephadex G75 column, and the eluate was monitored for Zn content and presence of -SH groups. A low molecular weight Zn peak was identified as an MT on the basis of Mr value and -SH/Zn ratio, using as a reference standard authentic horse kidney MT. Extracts prepared from embryos at different stages were analyzed by gel filtration chromatography. The results showed that the MT content decreased during cleavage up to the swimming blastula stage, and raised again at the early gastrula stage. In order to study MT synthesis during development, embryos were pulse-labelled in vivo with cysteine "S. The results showed that the change of MT synthesis paralleled that of the MT content. These data show that in the sea urchin embryo MT synthesis is differentially regulated.
P376 IDENTIFICATION OF Cd+-DEPENDENT CAMS IN XENOPUS DEVELOPMENT BY USE OF INTERSPECIES HOMOLOGY Frank Herzbarg, Annette P6ting and Doris Wedlich. lnstitut fiir Molekularbiologie und Biochemie. Freie UniversiM Berlin, Arnimallee 22, 1000 Berlin 33, West Germany Ca2+-dependent cell adhesion molecules (CAMS) are transmembrane glycoproteins structurally and functionally related in mammalian and avian species, suggesting that Ca2+-dependent CAMs consist of an evolutionary conserved gene family. Antibodies or cDNA probes specific either to the extracellular part or the cytoplasmic domain of uvomorulin were compared for their ability to detect corresponding molecules in Xenopus. Only antibodies directed against the evolutionary highly conserved cytoplasmic domain provided a distinct membrane staining on sections of Xenopus embryos or on cultured Xenopus epithelial cells by immunofluorescence. However, these antibodies recognized different polypeptides of 158, 140 and 128 kd in immunoblots prepared from Con A-Sepharose concentrated cell lysates of epithelial, neural, muscle and embryonic tissues. In concordance with the antibody analysis, signals in Northern hybridizations were only obtained when the cDNA probe encoding the cytoplasmic domain of uvomorulin was used. This probe hybridized with different mRNA species of 4.3, 4.1, 3.8 and 3.2 kb in various tissues. These results provide further evidence that the Ca2+-dependent CAMs are evolutionary conserved. The variety of polypeptides and transcripts observed in Xenopus indicates that several mambsrs of this gene family were detected by the use of probes specific lo conserved sequences. More important. with this approach we also identified molecules of this gene family in early developmental stages of Xenopus. Since these proteins wera present in mature eggs but not in oocytes, we assume a maternal store of Ca2+-dependent CAM RNA6 whose translation might be initiated during egg maturation.
172
THE CHOLINERGIC GASTRULATING
P373
CHICK
PROPERTIES ENGRYO
OF
THE
Tiit Laasherg, Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 200026, Estonia The gastrulating chick embryo has the main cholinergic characteristics whose acetylcholinpresence is indicated by esterase (AChE, EC 3.1.1.7) and choline acetyltransferase (CAT, EC 2.3.1.6) activand by the existence of functionally ity, active muscarinic receptors which are phosphatidylinositol turncoupled with over. A sharp increase in AChE activity occured in the gastrulating embryo. The highest activity was associated with hypoblast cells. By sucrose density gradient centrifugation three molecular forms of AChE with sedimentation coefficients 4.7S, 6.8s and 10.9s were determined. During the gastrulation there was no remarkable change in the activity of CAT. concentration The intracellular of acetylcholine (ACh) in gastrulating chick
embryo cells was about 10-6-10-5 M. ACh, via muscarinic receptors increased the from level of free calcium (tCa2+li) the basal level of 120 nM to the 200 nM (measand the level of InsP3. ured with indo-l), The establishment of dose-response curve by the measurement of [Ca2+Ii mobilization in dissociated embryo stages 3 and 4-5 by Hamburger (1951) gave the ED50 value for * 10-e M.
cells at and Hamilton ACh 4cLO.5)
STEADY IONIC CURRENTS AROUND THE UNFERTILISED EGG OF THE SEA URCHIN, HOLOPNEUSTES PURPURESCENS.
P375
Jane M. Chrystal, Robyn L. Overall and Valerie B. Morris. School of Biological Sciences, University of Sydney, Australia, 2006. A substantial part of our understanding of early animal
development
comes
from
studies
of
sea urchin
eggs and embryos. To date, there have been no reports of sea urchin eggs having steady ionic currento those reported for plant and other ts. similar animal embryos, where the currents have been shown to have developmental significance (Jaffe, 1981). The lack of such reports is probably because the sea urchins studied in North America and Europe have small eggs in the range, 40-lOOurn, that would make the measurement of steady ionic currents difficult. An Australian sea urchin, Holopneustes purpurescens, is a direct developing sea urchin with eggs approximately 400um in diameter. Their especially large size has opened the way for a study of steady ionic currents in sea urchins. Steady ionic currents were measured around these
eggs using Nuccitelii,
the vibrating 1974).
Steady
probe apparatus ionic
currents
(Jaffe
and
up to
2.4uA/cm were measured around the unfertilised eggs of Ii. purpurescens. The pattern of currents tend to be aroused as areas of inward or outward currents. ?he patterns are not mosaics. The data fits a model in which current enters half the egg The current patterns and leaves the other half. appear not to be associated with the area of grey pigmentation of the eggs or with the orientation of The ability to the epg with respect to gravity. measure steady ionic currents is limited by a low signal to noise ratio caused by the high conductivity of seawater. Ours is the first report of the measurement steady ionic currents around a marine
animal.
international
Reports,
Vol. 14, Abstracts
Supplement
1990
CHANGESOFMET~IONEIN CONTRN!l! IN SEAURCHINEMBRYOS B. De Petrocellis, P. De C. Capasso, Prisco, E. Gargiulo, E. Parisi, R. Scudiero. CM Institute of Protein Biochemistry and Enzymology, Zoological Station, 80121 Napoli, Italy
P374
We have studied the changes of the metallothionein (MT) content during development of the sea urchin egg. An extract prepared from unfertilized eggs was fractionated by gel filtration chromatography on a Sephadex G75 column, and the eluate was monitored for Zn content and presence of -SH groups. A low molecular weight Zn peak was identified as an MT on the basis of Mr value and -SH/Zn ratio, using as a reference standard authentic horse kidney MT. Extracts prepared from embryos at different stages were analyzed by gel filtration chromatography. The results showed that the MT content decreased during cleavage up to the swimming blastula stage, and raised again at the early gastrula stage. In order to study MT synthesis during development, embryos were pulse-labelled in vivo with cysteine "S. The results showed that the change of MT synthesis paralleled that of the MT content. These data show that in the sea urchin embryo MT synthesis is differentially regulated.
P376 IDENTIFICATION OF Cd+-DEPENDENT CAMS IN XENOPUS DEVELOPMENT BY USE OF INTERSPECIES HOMOLOGY Frank Herzbarg, Annette P6ting and Doris Wedlich. lnstitut fiir Molekularbiologie und Biochemie. Freie UniversiM Berlin, Arnimallee 22, 1000 Berlin 33, West Germany Ca2+-dependent cell adhesion molecules (CAMS) are transmembrane glycoproteins structurally and functionally related in mammalian and avian species, suggesting that Ca2+-dependent CAMs consist of an evolutionary conserved gene family. Antibodies or cDNA probes specific either to the extracellular part or the cytoplasmic domain of uvomorulin were compared for their ability to detect corresponding molecules in Xenopus. Only antibodies directed against the evolutionary highly conserved cytoplasmic domain provided a distinct membrane staining on sections of Xenopus embryos or on cultured Xenopus epithelial cells by immunofluorescence. However, these antibodies recognized different polypeptides of 158, 140 and 128 kd in immunoblots prepared from Con A-Sepharose concentrated cell lysates of epithelial, neural, muscle and embryonic tissues. In concordance with the antibody analysis, signals in Northern hybridizations were only obtained when the cDNA probe encoding the cytoplasmic domain of uvomorulin was used. This probe hybridized with different mRNA species of 4.3, 4.1, 3.8 and 3.2 kb in various tissues. These results provide further evidence that the Ca2+-dependent CAMs are evolutionary conserved. The variety of polypeptides and transcripts observed in Xenopus indicates that several mambsrs of this gene family were detected by the use of probes specific lo conserved sequences. More important. with this approach we also identified molecules of this gene family in early developmental stages of Xenopus. Since these proteins wera present in mature eggs but not in oocytes, we assume a maternal store of Ca2+-dependent CAM RNA6 whose translation might be initiated during egg maturation.