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Gastrulation in the sea urchin larva studied by aid of time-lapse cinematography
Experimental
Cell Research, 10, 733-756 (1956)
BRIEF GASTRULATION
733
NOTES
IN THE SEA URCHIN LARVA STUDIED OF TIME-LAPSE CINEMATOGRAPHY T. GUSTAFSON
BY AID
and H. KINNANDER
The Wenner-Gren Institute of Experimental Biology, University of Stockholm, Sweden Received February
11, 1956
THE mechanism of gastrulation, notably that of invagination, continues to be an object of great interest. This problem has been subject to thorough descriptive and experimental analysis in the amphibians, where the mechanism appears to be rather complex. A simpler type of mechanism may be expected to occur in many invertebrates. A careful study of the morphology of gastrulation in some representatives from this category of animals is of fundamental importance to future experimental studies. By means of time-lapse cinematography each morphological change in the embryo can be registered during the process of gastrulation. The transparency of many marine larvae facilitates the use of this technique. However, the rapid swimming movements of this kind of embryos constitute a serious obstacle to time-lapse filming and makes it impossible to keep the embryos in a constant position. This explains why e.g. the sea urchin larva has so far not been thoroughly analyzed as to its post-hatching morphogenetic movements, although the segmentation of the sea urchin egg was studied already at the beginning of this century. During the past three years the authors worked out a method by means of which the swimming movements are eliminated and the larvae are kept in a constant position for at least 12 hours. When using this method which is especially adapted for sea urchin larvae, the further development of the larvae is not affected. The device for keeping the larvae in a constant position consists of a piece of nylon net inserted between the slide and the coverglass. The nylon threads were made rough by a precipitate of calcium carbonate and were thus rather similar to barbed wire. The cross section of the meshes was somewhat larger than the diameter of the blastula. When an egg suspension was placed on the net and the coverglass was placed in position, the larvae were individually caught in the meshes. The calcium carbonate crystals caused slight depressions in the walls of the larvae which were thus kept in a constant position without much interference in their ciliar acitivity. A slow current of water was continuously passed below the coverslip by means of filter paper strips, one of which was siphoning water from a cuvette to the right edge of the cover glass, the other from the left edge to an effluent beaker. The morphogenetic movements were registered by time-lapse cinematography with a frequency of one picture per ten or nineteen seconds. The temperature was about 22°C.
An analysis of 350 meter time-lapse film of gastrulating larvae of Psammechinus revealed that there are two main phases in invagination. In the first the
miliaris
45 - 563703
Experimental
Cell Research 10
734
F. Ghiretti and
V. D’Amelio
archenteron rudiment elongates to about I/& to 1/8 its final length. This phase of invagination is probably autonomous, i.e. essentially dependent on factors residing in the vegetal region itself. Invagination can thus be attained in an isolated vegetal fragment of a blastula [3]. After this phase of invagination the archenteron tip will more or less remain on the same level for some time, or slowly advance in the animal direction. During the second phase of invagination the process occurs at a considerably higher rate than during the first phase and is here caused by pseudopodia emitted by the archenteron tip and attaching themselves to the ectoderm. The archenteron tip is pulled in the animal direction by contractions of these pseudopodia. The role of the pseudopodia is evident from the fact that their appearance closely corresponds to the increased rate of invagination, and also that a break of the pseudopodia can be correlated with a retardation of invagination. Furthermore, a bend of the archenteron is preceded by an eccentric formation of the pseudopodia in the same direction. Sooner or later the pseudopodia forming cells themselves are extracted from the archenteron tip owing to the contractions of the pseudopodia, which may be explained by the fact that the intestine cannot respond to the contractions at a sufficient rate. The cells liberated are now called secondary mesenchyme cells. The connections between these cells and the archenteron tip may be completely or partially interrupted during extraction. In such cases the invagination ceases until new pseudopodia are formed. A detailed description with further data on the technical device for this study and on results obtained will be presented in a later publication [2]. After our paper [2] had been submitted for publication in this journal, an important paper on the mechanism of invagination [l] appeared. The data presented therein on the contractile elements connecting the archenteron tip with the ectoderm are also in good agreement with our own observations. REFERENCES 1. DAN, K. and OKAZAKI, K., Biol. Bull. 110, 29 (1956). 2. GUSTAFSON, T. and KINNANDER, H., Exptl. Cell Research (In press). 3. MOORE, A. R. and BURT, A. S., J. Exptl. Zoot. 82, 159 (1939).
THE
METABOLISM
OF PENTOSE PHOSPHATE SPERM AND EGGS F. GHIRETTI
Department
of Physiology,
IN SEA URCHIN
and V. D’AMELIOI &a&one Zoologica, Nales, Italy.
Received February
11, 19.56
T HE
mechanism of stepwise oxidation of glucose through the glucose-6-phosphate dehydrogenase shunt has recently been elucidated by the investigations of Racker
1 Fellow of the Centro di Biologia de1 Consiglio Istituto di Anatomia Comparata, Palermo. Experimental
Cell Research 10
Nazionale
delle Ricerche.
Present
address:
Cell Research, 10, 733-756 (1956)
BRIEF GASTRULATION
733
NOTES
IN THE SEA URCHIN LARVA STUDIED OF TIME-LAPSE CINEMATOGRAPHY T. GUSTAFSON
BY AID
and H. KINNANDER
The Wenner-Gren Institute of Experimental Biology, University of Stockholm, Sweden Received February
11, 1956
THE mechanism of gastrulation, notably that of invagination, continues to be an object of great interest. This problem has been subject to thorough descriptive and experimental analysis in the amphibians, where the mechanism appears to be rather complex. A simpler type of mechanism may be expected to occur in many invertebrates. A careful study of the morphology of gastrulation in some representatives from this category of animals is of fundamental importance to future experimental studies. By means of time-lapse cinematography each morphological change in the embryo can be registered during the process of gastrulation. The transparency of many marine larvae facilitates the use of this technique. However, the rapid swimming movements of this kind of embryos constitute a serious obstacle to time-lapse filming and makes it impossible to keep the embryos in a constant position. This explains why e.g. the sea urchin larva has so far not been thoroughly analyzed as to its post-hatching morphogenetic movements, although the segmentation of the sea urchin egg was studied already at the beginning of this century. During the past three years the authors worked out a method by means of which the swimming movements are eliminated and the larvae are kept in a constant position for at least 12 hours. When using this method which is especially adapted for sea urchin larvae, the further development of the larvae is not affected. The device for keeping the larvae in a constant position consists of a piece of nylon net inserted between the slide and the coverglass. The nylon threads were made rough by a precipitate of calcium carbonate and were thus rather similar to barbed wire. The cross section of the meshes was somewhat larger than the diameter of the blastula. When an egg suspension was placed on the net and the coverglass was placed in position, the larvae were individually caught in the meshes. The calcium carbonate crystals caused slight depressions in the walls of the larvae which were thus kept in a constant position without much interference in their ciliar acitivity. A slow current of water was continuously passed below the coverslip by means of filter paper strips, one of which was siphoning water from a cuvette to the right edge of the cover glass, the other from the left edge to an effluent beaker. The morphogenetic movements were registered by time-lapse cinematography with a frequency of one picture per ten or nineteen seconds. The temperature was about 22°C.
An analysis of 350 meter time-lapse film of gastrulating larvae of Psammechinus revealed that there are two main phases in invagination. In the first the
miliaris
45 - 563703
Experimental
Cell Research 10
734
F. Ghiretti and
V. D’Amelio
archenteron rudiment elongates to about I/& to 1/8 its final length. This phase of invagination is probably autonomous, i.e. essentially dependent on factors residing in the vegetal region itself. Invagination can thus be attained in an isolated vegetal fragment of a blastula [3]. After this phase of invagination the archenteron tip will more or less remain on the same level for some time, or slowly advance in the animal direction. During the second phase of invagination the process occurs at a considerably higher rate than during the first phase and is here caused by pseudopodia emitted by the archenteron tip and attaching themselves to the ectoderm. The archenteron tip is pulled in the animal direction by contractions of these pseudopodia. The role of the pseudopodia is evident from the fact that their appearance closely corresponds to the increased rate of invagination, and also that a break of the pseudopodia can be correlated with a retardation of invagination. Furthermore, a bend of the archenteron is preceded by an eccentric formation of the pseudopodia in the same direction. Sooner or later the pseudopodia forming cells themselves are extracted from the archenteron tip owing to the contractions of the pseudopodia, which may be explained by the fact that the intestine cannot respond to the contractions at a sufficient rate. The cells liberated are now called secondary mesenchyme cells. The connections between these cells and the archenteron tip may be completely or partially interrupted during extraction. In such cases the invagination ceases until new pseudopodia are formed. A detailed description with further data on the technical device for this study and on results obtained will be presented in a later publication [2]. After our paper [2] had been submitted for publication in this journal, an important paper on the mechanism of invagination [l] appeared. The data presented therein on the contractile elements connecting the archenteron tip with the ectoderm are also in good agreement with our own observations. REFERENCES 1. DAN, K. and OKAZAKI, K., Biol. Bull. 110, 29 (1956). 2. GUSTAFSON, T. and KINNANDER, H., Exptl. Cell Research (In press). 3. MOORE, A. R. and BURT, A. S., J. Exptl. Zoot. 82, 159 (1939).
THE
METABOLISM
OF PENTOSE PHOSPHATE SPERM AND EGGS F. GHIRETTI
Department
of Physiology,
IN SEA URCHIN
and V. D’AMELIOI &a&one Zoologica, Nales, Italy.
Received February
11, 19.56
T HE
mechanism of stepwise oxidation of glucose through the glucose-6-phosphate dehydrogenase shunt has recently been elucidated by the investigations of Racker
1 Fellow of the Centro di Biologia de1 Consiglio Istituto di Anatomia Comparata, Palermo. Experimental
Cell Research 10
Nazionale
delle Ricerche.
Present
address: