Properties and functions of the surface coat in amphibian embryos

High molecular weight DNA was isolated from the yolk platelets of Xenopus laevis oocytes ovulated in vitro. Yolk DNA has the same buoyant density in CsCl gradients as mitochondrial and nuclear DNAs, and, like them, it is double-stranded. Yolk DNA behaves like nuclear DNA, and not like mitochondrial DNA, upon renaturation after denaturation. The molecules are always linear. Cytochemical and biochemical controls preclude the possibility that the yolk DNA might be contaminated by nuclear or mitochondrial DNA. We conclude that the yolk DNA is an endogenous component of the yolk platelets.As described in a previous paper (1), DNA could be extracted from the yolk platelets isolated from whole ovaries of Xenopus laevis. Since it was likely that the yolk fraction was contaminated with nuclear DNA from the follicle cells in these earlier experiments, we now worked with oocytes that have been freed from their follicle cells in two differentways: (i) manual removal of follicle cells after a short treatment of the oocytes with chemical agents, such as Pronase (0.15%) or sodium dodecyl sulfate (0.3%); (ii) in vitro ovulation of the oocytes in the presence of hormones. In both cases, high molecular weight DNA could be extracted from the yolk platelets; the physicochemical properties of yolk DNA from oocytes ovulated in vitro will be described. Our experimental data exclude beyond reasonable doubt the possible contamination of yolk DNA by either nuclear or mitochondrial DNA arising from follicular or other ovarian contaminating cells. We shall discuss the extraction of yolk DNA from unfertilized eggs of Xenopus and Ambystoma mexicanum. MATERIALS AND METHODSIn Vitro Ovulation. Ovaries were removed from adult Xenopus laevis, and the membranes of the ovarian follicles were torn apart. (ViPyr). (pH 7.0); 10 ml of medium were used for each 1000 oocytes. The homogenate was layered on an equal volume of 1 M sucrose-5% (ViPyr). and centrifuged for 20 min at 500 X g. The pellet was suspended in 0.25 M sucrose-5% (ViPyr). and centrifuged again in the same way. Electron microscopic controls showed that contamination of the yolk platelets by pigment and mitochondria was negligible.Mitochondria were isolated by Dawid's method (4). Removal of the Jelly of Unfertilized Shed Eggs. For Xenopus, a cysteine-papain mixture at pH 7.8 was used (4). For Ambystoma, the jelly was removed with forceps without any chemical treatment. Both types of eggs were maintained in 0.1 X Holtfreter's medium (5).DNA Determination. Instead of the fluorometric method (6), we used a modification of Keck's indol method (7): 75 oocytes were homogenized in 2 ml of 0.25 M sucrose-0.03 M Tris HCl (pH 7.4)-0.1 M EDTA; 2 ml of cold 10% Cls-CCOOH were added; after 15 min, the pellet was centrifuged and washed with cold 5% Cl3CCOOH. Lipids were removed by single successive washings with 94% alcohol at 40, 94% alcohol at 500, n-butyl alcohol at 500, alcohol-ether (3: 1) at 500, and ether. The pellet was extracted for 20 min at 1000 with 600 ,l of 5% Cl3CCOOH and centri...

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“…These two hormonal treatments are known to be most efficient in inducing, respectively, maturation and ovulation (2). After 5 Taken from ref. 6.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Yolk DNA from Xenopus laevis Oocytes Ovulated In Vitro

Hanocq

Kirsch‐Volders

Hanocq-Quertier

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

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“…As in the present experiments with zebrafish tissues, frog (deep) mesoderm or endoderm enveloped (deep) ectoderm in vitro. The key to understanding the reverse of this sequence in the intact embryo was provided by Holtfreter (1943), who showed that the apical surfaces of the cells of the amphibian blastula are nonadhesive to other cells. He called this apical region the "surface coat" and showed that cells bearing it are forced to lie at free surfaces like those facing the outside or the archenteron.…”

Section: Surface Tensions and Normal Germ Layer Arrangementmentioning

confidence: 99%

Quantitative differences in tissue surface tension influence zebrafish germ layer positioning

et al. 2008

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“…Although transcellular currents were indeed measured, their role in the maintenance of apical/basolateral polarity is still unclear (Kline et al, 1983;Nuticelli and Wiley, 1985). Holtfreter (1943aHoltfreter ( , 1948 originally inferred that polarity of superficial blastomeres is maintained because of the presence of extracellular deposits, the "surface coat." We have found that the apical membrane domain of 1/64 cells harbours a dense glycocalyx, which may be related to the "surface coat."…”

Section: Maintenance Of Agicaubasolateral Polarity Of Blastomeres In mentioning

confidence: 99%

“…Accordingly, the cells on the surface of a Xenopus blastula are joined by epithelial cell junctions and exhibit the apical/basolateral membrane polarity typical for epithelial cells (Holtfreter, 1943a(Holtfreter, ,b, 1948Sanders and Zalik, 1972;Armstrong et al, 1982;this communication). The outer, apical membrane domains of these cells originate from egg membrane and are nonadhesive (Holtfreter, 1943a,b;Byers and Armstrong, 1986).…”

mentioning

confidence: 98%

Epithelial cell polarity in early Xenopus development

Müller

Hausen

1995

Developmental Dynamics

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The Xenopus blastula consists of two morphologically distinct cell types. Polarized epithelial cells build up the embryonic surface and fence off an inner non-polarized cell population. We examined the establishment of this early functional cell diversification in the embryo by single cell analysis, in vitro cell culture, and transplantation experiments. Single blastomeres from a 64-cell embryo (1/64 cells) exhibit several features of polarized cells. The plasma membrane of 1/64 cells consists of an apical domain, which is inherited from the original egg membrane, and a basolateral domain derived from newly formed membrane during cleavage. These are inherent, cell-autonomous properties of the blastomeres, as they form and are maintained in blastomeres raised in the absence of any cell interactions in calcium free medium. Upon in vitro culture a single 1/64 cell gives rise to an aggregate of two different cell types. Cells carrying a part of the former egg membrane domain differentiate into polarized epithelial cells, whereas cells lacking this membrane domain are not polarized. These results demonstrate that the inclusion of the egg membrane, rather than external signals related to the position of a cell in the intact embryo, is required for the apicalhasolateral differentiation of the surface epithelium. This view is supported by cell transplantation studies. A single 1/64 cell was implanted into the blastocoel of a stage 8 blastula embryo. The progeny of the implanted cell proliferate within the host embryo and split into two morphologically distinct populations with different cell behaviours. Cells incorporating a part of the egg membrane form coherent patches of polarized epithelial cell sheets in the interior of the host embryo. In contrast, cells lacking egg membrane do not exhibit any characteristics of polarized cells and eventually spread into different regions of the host embryo. Our results show that the egg membrane and/or components of the submembrane cortex play a determinative role in the formation of the blastula epithelium.

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Properties and functions of the surface coat in amphibian embryos

Cited by 312 publications

References 60 publications

Characterization of Yolk DNA from Xenopus laevis Oocytes Ovulated In Vitro

Characterization of Yolk DNA from Xenopus laevis Oocytes Ovulated In Vitro

Quantitative differences in tissue surface tension influence zebrafish germ layer positioning

Epithelial cell polarity in early Xenopus development

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