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Quail-chick chimeras to study neural and immune system development
344
343
Invagination of the Neural Plate through the Blastopore in Urodele A~phibian
OUAIL-CHICK CHI~IERAS TO STUDY NEURAL AND IMMUNE SYSTEM DEVELOPMENT. C. ~!artin, M.A. T e i l l e t , H. Ohki and N. Le Douarin. I n s t i t u t d'Embryologie c e l l u l a i r e et mol~cul a i r e du CNRS-Nogent-sur-Marne (France).
Osamu N~
He present a videotape where microsuroical operations are perfomed on quail and chick embryos. For example on two day old chick embryos, a piece of the neural tube or of the brain anlagen can be s e l e c t i v e l y removed and replaced by t h e i r quail counterpart. These neural chimeras are able to hatch and are prQdous tools to study certain aspects of the neural development due to the nuclear marker carried by quail c e l l s . Other chimeras are also s u r g i c a l l y constructed between 4 or 5 day old chick and quail embryos in view of studying the development of the immune swstem and the mechanisms through which tolerance to s e l f is induced during ontogeny. A quail wing bud grafted on a chick r e c i p i e n t at E5 develop normally during embryonic l i f e but is acutely rejected a f t e r b i r t h . He showed that i f double t r a n s p l a n t a t i o n of the winn and of thymic epithelium rudiment from the same donor is carried out at E4 permanent tolerance of the wing is induced. This demonstrates the role of the epithelium of the thymus in the establishment of tolerance to s e l f .
Fifty years ago, in 1938, I presented a new fate-map of the amphibian gastrula,guite different from vogt' s~ classical one. Soon after, Pasteels (1939)}verifie(1 my map and applied it to european species. My map was drawn basing on two important ~indings by myself;(1 )the posterior part of the neural plate is occupied by the material of scmites which Originates from the dorsomedian portion of the early gastrula, (2) the posterior half of this part is tacked and rolled in over the rim of slit-shaped blastopore. The movement is continued until the neural folds fuse to form a tube. The material invaginated by this movement forms scmites in the posterior trunk region. Only the first point of my findings is known in Europe, while the second point has not yet noted so well. In my original research, the movement in guestion was proved by tracing displacement of stained ~arks in successive stages. Now, the same movement is shown directly to the audience by the visual demonstration with the aid of videocamera.
345
346
OOPLASMIC
MOVEMENTS
AND
CALCIUM
WAVES
A NEW TECHNIQUE RAT EMBRYOS
AT
FERTILIZATION IN ASCIDIAN EOOS J.E. Speksnijder, C. Sardet, L.F. Jaffa, and S.I. Inou4; Dept. Exp. Zoolo~, University of Utrecht, The N e t h e r l a n d s , and Marine B i o l o & i c a l L a b o r a t o r y , Woods Hole, MA. USA.
FOR IN VIVO EXPERIMENTS
Van der Zee D9 I, VermeiJ-Keers van Prooije A E
IN
Chr I, Smlts-
i Dept. of Anat., Leiden, The Netherlands, 2 TNO, Zeist, The Netherlands
Using various imaging techniques, we have studied the relationship between the ooplasmic movements and the waves of elevated free calcium that follow fertilization in the extremely clear eggs of the asicidian Phallusia mammillata. Time-lapse video microscopy using rectified differential interference contrast and polarization optics show the cortical contraction and the proces of ooplasmic segregation in great detail. Simultaneous imaging of both the transmitted type image and the calcium-dependent luminescence of aequorin-loaded eggs was made possible using a 650 nm cut-off filter and a red sensitive Newvicon tube to record the transmitted image in the red part of the spectrum, and a dichroic mirror to reflect the bluish aequorin light to an Imaging Photon Detector. Overlaying these two images revealed that 1) the calcium wave starts at the point of sperm entry and preceeds the cortical contraction wave by about q5 sac; 2) the fertillzation pulse is followed by a series of calcium oscillations that also travel as waves with the majority starting near the vegetal pole; 3) these calcium waves usualy preceed a contraction of the egg surface in animal direction; and g) the last few pulses in the series are frequently followed by an "echo" wave originating in the animal hemisphere.
The in vitro culture of whole mammalian embryos can only be used for experiments at early developmental stages, ~.g. in rat embryos of 8.5 to 13 days post coltum at the most, and is limited to 48 hours. After two days of culture, symptoms of generalized cell degeneration are interfering with the results of the experiments. Up to now in vivo manipulation in rat embryos of 15 days post coitum and younger could not be performed. Therefore, a new in utero operation technique has been developed by using an endoscope. In vivo manipulations, e.g. microinJectlons, in embryos of 11.5-12 days o f gestation can now be carried out under endoscopic quidance. As a consequence, the results of the experiments can be followed until term or even thereafter. A video film will present this technique.
S102
343
Invagination of the Neural Plate through the Blastopore in Urodele A~phibian
OUAIL-CHICK CHI~IERAS TO STUDY NEURAL AND IMMUNE SYSTEM DEVELOPMENT. C. ~!artin, M.A. T e i l l e t , H. Ohki and N. Le Douarin. I n s t i t u t d'Embryologie c e l l u l a i r e et mol~cul a i r e du CNRS-Nogent-sur-Marne (France).
Osamu N~
He present a videotape where microsuroical operations are perfomed on quail and chick embryos. For example on two day old chick embryos, a piece of the neural tube or of the brain anlagen can be s e l e c t i v e l y removed and replaced by t h e i r quail counterpart. These neural chimeras are able to hatch and are prQdous tools to study certain aspects of the neural development due to the nuclear marker carried by quail c e l l s . Other chimeras are also s u r g i c a l l y constructed between 4 or 5 day old chick and quail embryos in view of studying the development of the immune swstem and the mechanisms through which tolerance to s e l f is induced during ontogeny. A quail wing bud grafted on a chick r e c i p i e n t at E5 develop normally during embryonic l i f e but is acutely rejected a f t e r b i r t h . He showed that i f double t r a n s p l a n t a t i o n of the winn and of thymic epithelium rudiment from the same donor is carried out at E4 permanent tolerance of the wing is induced. This demonstrates the role of the epithelium of the thymus in the establishment of tolerance to s e l f .
Fifty years ago, in 1938, I presented a new fate-map of the amphibian gastrula,guite different from vogt' s~ classical one. Soon after, Pasteels (1939)}verifie(1 my map and applied it to european species. My map was drawn basing on two important ~indings by myself;(1 )the posterior part of the neural plate is occupied by the material of scmites which Originates from the dorsomedian portion of the early gastrula, (2) the posterior half of this part is tacked and rolled in over the rim of slit-shaped blastopore. The movement is continued until the neural folds fuse to form a tube. The material invaginated by this movement forms scmites in the posterior trunk region. Only the first point of my findings is known in Europe, while the second point has not yet noted so well. In my original research, the movement in guestion was proved by tracing displacement of stained ~arks in successive stages. Now, the same movement is shown directly to the audience by the visual demonstration with the aid of videocamera.
345
346
OOPLASMIC
MOVEMENTS
AND
CALCIUM
WAVES
A NEW TECHNIQUE RAT EMBRYOS
AT
FERTILIZATION IN ASCIDIAN EOOS J.E. Speksnijder, C. Sardet, L.F. Jaffa, and S.I. Inou4; Dept. Exp. Zoolo~, University of Utrecht, The N e t h e r l a n d s , and Marine B i o l o & i c a l L a b o r a t o r y , Woods Hole, MA. USA.
FOR IN VIVO EXPERIMENTS
Van der Zee D9 I, VermeiJ-Keers van Prooije A E
IN
Chr I, Smlts-
i Dept. of Anat., Leiden, The Netherlands, 2 TNO, Zeist, The Netherlands
Using various imaging techniques, we have studied the relationship between the ooplasmic movements and the waves of elevated free calcium that follow fertilization in the extremely clear eggs of the asicidian Phallusia mammillata. Time-lapse video microscopy using rectified differential interference contrast and polarization optics show the cortical contraction and the proces of ooplasmic segregation in great detail. Simultaneous imaging of both the transmitted type image and the calcium-dependent luminescence of aequorin-loaded eggs was made possible using a 650 nm cut-off filter and a red sensitive Newvicon tube to record the transmitted image in the red part of the spectrum, and a dichroic mirror to reflect the bluish aequorin light to an Imaging Photon Detector. Overlaying these two images revealed that 1) the calcium wave starts at the point of sperm entry and preceeds the cortical contraction wave by about q5 sac; 2) the fertillzation pulse is followed by a series of calcium oscillations that also travel as waves with the majority starting near the vegetal pole; 3) these calcium waves usualy preceed a contraction of the egg surface in animal direction; and g) the last few pulses in the series are frequently followed by an "echo" wave originating in the animal hemisphere.
The in vitro culture of whole mammalian embryos can only be used for experiments at early developmental stages, ~.g. in rat embryos of 8.5 to 13 days post coltum at the most, and is limited to 48 hours. After two days of culture, symptoms of generalized cell degeneration are interfering with the results of the experiments. Up to now in vivo manipulation in rat embryos of 15 days post coitum and younger could not be performed. Therefore, a new in utero operation technique has been developed by using an endoscope. In vivo manipulations, e.g. microinJectlons, in embryos of 11.5-12 days o f gestation can now be carried out under endoscopic quidance. As a consequence, the results of the experiments can be followed until term or even thereafter. A video film will present this technique.
S102