- Email: [email protected]
Electron probe analysis of calcium distribution during active transport in chick chorioallantoic membrane
216
J. R. Coleman et al.
permit the complete separation of chromosomes of one specific size from other chromosomes. Perhaps this difficulty could be resolved by using cell types in which there is a real diversity in chromosome size. The spectrum of morphological diversity among the 44 chromosomes of the Syrian hamster complement is greater than that in either the HeLa cell or L cell complements. However, even the fractionation of these chromosomesdid not result in a greatly improved separation of individual types of chromosomes from one another. Better success might be obtained by using cell types such as those from Microtus agrestis, in which there is an extreme size difference between the X chromosomes and the autosomes. This work forms part of a doctoral dissertation submitted by G. D. Burkholder to the Faculty of Graduate Studies and Research, McGill University. The investigation was supported by Grant MT2169 from the Medical Research Council of Canada, and a postgraduate scholarship from the National Research Council of Canada.
REFERENCES 1. Burkholder, G D & Mukherjee, B B, Exptl cell res 61 (1970) 413. 2. Huberman, J A & Attardi, G, J cell biol 31 (1966) 95. 3. 1 J mol biol 29 (1967) 487. 4. Maio, J J & Schildkraut. C L. J mol biol 24 (1967) 29. 5. - Ibid 40 (1969) 203. 6. Matsuya, Y & Yamane, I, Exptl cell res 50 (1968) 652. 7. Mendelsohn, J, Moore, D E & Salzman, N P, J mol biol 32 (1968) 101. 8. Robbins, E & Marcus, P I, Science 144 (1964) 1152. 9. Salzman, N P, Moore, D E & Mendelsohn, J, Proc natl acad sci US 56 (1966) 1449. 10. Schneider, E L & Salzman, N P, Science 167 (1970) 1141. 11. Terasima, T & Tolmach, L J, Exptl cell res 30 (1963) 344. Received June 25, 1970
Exptl Cell Res 63
Electron probe analysis of calcium distribution during active transport in chick chorioallantoic membrane J. R. COLEMAN, S. M. DeWITT, P. BATT and A. R. TEREPKA, Department of Radiation Biology and Biophysics, University of Rochester, School of Medicine and Dentistry, Rochester, N. Y. 14620, USA Summary The embryonic chick chorioallantoic membrane actively transports calcium and the transported calcium is compartmentalized or sequestered, since it does not mix completely with calcium already contained within the membrane. Correlated electron probe and electron microscope analysis shows that only certain ectodermal cells of the membrane contain concentrations of calcium. We suggest that these are specialized cells which are specifically involved in trans-epithelial transport of calcium and represent containing the sequestered the “compartments” calcium.
The ectoderm of the embryonic chick chorioallantoic membrane (CAM) develops the ability to transport calcium against an electrochemical gradient after about 13-14 days of egg incubation [l], a time which corresponds with the rapid movement of calcium from the eggshell to the embryo for skeletal calcification [2]. Transport chamber studies of the isolated membrane using 45Casuggestedthat the calcium being transported through the membrane was compartmentalized [ 11.Even after 6 h of exposure to 45Ca,the spec. act. of the membrane was only about twothirds that of the outside bathing solution. However, the spec.act. of the calcium transported to the inside solution was significantly higher than that of the membrane and close to that of the outside solution from which the calcium was transported. The ectoderm of the CAM is an epithelium forming a continuous layer of cells under the non-cellular egg shell membranes. It is composed of at least three different cell types supporting and surrounding respiratory capillaries [3]. The “compartment” sequestering calcium in transit could be restricted to a
Calcium in chorioallantoic membrane
certain type of cell in the epithelium or could be a separate compartment within every cell. To help distinguish between these two alternatives, we used the electron probe X-ray microanalyzer to localize sites of calcium concentration within the CAM. If the distribution of calcium found in the CAM were homogeneous, it would indicate that each cell contains a “compartment” for the calcium in transport. The electron probe revealed, however, that the calcium was concentrated only within certain cells, suggesting that these specialized epithelial cells were responsible for the active calcium transport by this membrane. Material and Methods Tissues were fixed for 30 min to 1 h with 6% acrolein (synthesized by Dr W. G. Aldridge, University of Rochester) in 0.1 M cacodylate buffer (pH 7.2) containing 1% sodium oxalate. This was followed by a short wash in the buffer, a 30 min postfixation with 1% 0~0, in the same buffer, dehydration in ethanol and embedding in Araldite@ or paraffin. Preliminary studies had shown that this procedure produced no substantial loss of total calcium or ““Ca and provided good electron microscope morphology. Two-micron thick sections of CAM were cut without exposure to water, mounted on silicon wafers [5] dry or after expansion on 5 % sodium oxalate at 60°C and coated lightly with vacuumevaporated aluminium or carbon after removal of paraffin with xylene or benzene. These were examined with an Applied Research Laboratory Electron Probe X-ray M&oanalyzer, EMX, usually at 22 kV accelerating potential and about 7 x lo-+’ A sample current, with a beam less than 1 pm in diameter. Images were recorded from the oscilloscope screen on Polaroid@ film. The CAM was removed from the incubated egg and mounted in an Ussing-type transport chamber. Oxygenated Krebs-Ringer bicarbonate solution containing 1.0 mM calcium was circulated on both sides and 46Ca was added to the outside (ectoderm) solution. Samples from the inside and outside solutions were taken at intervals and assayed for total calcium and 46Ca to insure that the CAM was actively transporting calcium [l]. The membrane was then removed from the chamber, fixed, dehydrated, embedded, sectioned and examined with the probe as described above. Adjacent, or nearly adjacent, thin sections of Araldite@ embedded tissues were sectioned and prepared in a conventional manner for electron microscopy. Specimens of CAM were prepared from eggs incubated for at least 14 days and known to be actively transporting calcium, and from eggs incubated less
217
than 13 days so that the active transport process had not started. Also examined were- membranes in which calcium transport was inhibited by incubation in a nitrogen atmosphere and mature-membranes exposed to a solution containing strontium which is also actively transported but at a rate about onethird that of calcium [l].
Results
Electron probe analysis showed that discrete calcium localizations occurred only in the ectodermal cell layer in mature, transporting CAM (fig. 1a). The corresponding sample current image of this tissue section is shown in fig. 1 b. Phosphorus and sulfur scans of the specimen (fig. 1 c, d) showed generalized distributions which agree with the known chemical concentrations of these elements. The calcium concentrations visualized with the electron probe appeared to be limited to certain ectodermal cells which were found immediately adjacent to respiratory capillaries in the corresponding sample-current images (fig. 2a, b). These cells could be identified in the electron microscope as having long cytoplasmic processes extending between the endothelial cells of capillaries and the shell membrane (fig. 2 c). The cytoplasmic processesthat lay between the capillaries and the shell membrane contain 400 to 1 000 A diameter vesicles. The mitochondria of these cells cluster around the nucleus which is usually located near the basal surface and some distance from the calciumcontaining cytoplasmic processes adjacent to the blood capillaries. No discrete calcium localizations were found with the electron probe in young, non-transporting membranes or in membranes that had been incubated in nitrogen, even though both of these membranes had been exposed to the same concentration of calcium as the mature, transporting membranes. When a mature, transporting membrane was exposed to strontium rather than calcium, strontium localizations were found at Exptl Cell Res 63
218
J. R. Coleman et al.
Fig. 1. u-d are X-ray and sample current images of the same section of chorioallantoic membrane (CAM) in the electron probe at a magnification of 10 ,um per screen division. The X-ray images are the result of long photographic integrations (40 min). Signals not associated with tissue components and randomly distributed through the background are due to noise and cannot be ascribed to the presence of any particular element. (a) Calcium X-ray image of CAM section showing calcium restricted to discrete sites within the membrane. The exact location of these can be determined by referring to the grid coordinates on the sample current image in 1b. (6) Sample current image showing shell membrane (SM) above the respiratory capillaries which appear as dark areas in the ectodermal cell layer (EC). A large blood vessel (BP) containing red blood cells is in the mesoderm (M) and the thin endodermal layer (EN) is seen near the bottom of the picture. Three sites of calcium localization are marked by arrows; the others can be determined from coordinates of grid lines in the calcium X-ray image. (c) Sulfur X-ray image of same section showing concentration of sulfur mainly restricted to noncellular shell membrane (SM). Note that sulfur signal is not restricted to discrete sites as is calcium. (6) Phosphorus X-ray image of same section showing concentration of phosphorus reflecting distribution of cellular material throughout the CAM.
the same sites as calcium. Since membranes not exposed to strontium experimentally had no detectable strontium by probe analysis, this indicates that the observed concentration sites contained the divalent ions which were being actively transported by the membrane. The validity of these electron probe analyses, as with all histochemical techniques, is dependent upon tissue preparation proceExptl Cell Res 63
dures that neither lose nor redistribute calcium and yet preserve cell morphology. In the studies reported here, calcium loss was assessedby incubating mature, transporting membranes with 45Caas described [I], then dividing the membrane in two. One-half was analyzed directly for total calcium and 45Ca and the second half was analyzed after being subjected to the fixing and embedding proce-
Calcium in chorioallantoic membrane 219
Fig. 2. a and b are, respectively, sample current image and the corresponding calcium X-ray image of the same section at a magnification of 5 pm/grid square. In (a) portions of the shell membrane (S&f), ectoderm (E) and mesoderm (M) are visible. Directly beneath the shell membrane: ectoderm junction are capillaries which appear as dark spaces and contain three bright, roughly disc-shaped erythrocytes, one of which is labelled R. Between the two capillaries is an ectodermal cell (similar to that seen between two capillaries in (c) which extends from the interior of the ecoderm to the shell membrane and lies between the endothelial lining of the capillaries and the shell membrane. In (b) only a small discrete region gives evidence of calcium signals above background levels. This corresponds to a portion of the ectodermal cell seen between the two capillaries in (A) and is restricted to the region of the cell near the shell membrane. (c) Electron micrograph of thin section of CAM in a region similar to that seen in (a) showing the type of ectodermal cell seen between two capillaries. The ectodermal cell has long thin cytoplasmic processes that extend between the noncellular shell membrane (SW) and the endothelial cells of the capillaries (~5).These ectodermal cells are the sites of calcium concentration as determined by electron probe analysis. (Bar represents 1 ym.)
Exptl Cell Res 63
220
T. P. Lin & Joan Florence
dures. Total calcium content as well as the spec. act, of both halves were comparable. While no independent method to assess redistribution exists, we took the following indirect evidence to suggest that redistribution, if it occurred, was minimal, and was beyond the resolution of the electron probe: (1) When conditions which were likely to cause redistribution (e.g. distortion and destruction of cell morphology, floating sections on water) were employed, only a generalized distribution of calcium was observed. (2) Calcium localizations were found only in mature, transporting membranes. Significantly, these were not randomly distributed but were associatedwith a particular cell type occupying a characteristic site within the ectoderm. Electron microscope examination of these cells gave no indication of crystalline deposits. (3) Calcium chloride ranging x 100 above and below the amount found in membranes was dissolved in 10% albumen which was then processedby the methods indicated. No discrete localizations were found and calcium remained homogeneously distributed. In conclusion, we suggestthat the compartment which sequesterscalcium and prevents it from mixing with calcium already within the membrane is restricted to certain ectodermal cells which are specifically involved in active calcium transport. These cells surround the respiratory capillaries with long cytoplasmic processes. Such a juxtaposition would facilitate the movement of eggshell calcium into the embryo’s blood plasma. Whether the vesicles noted in the cells that contain calcium localizations are directly involved with the transport of calcium [l] remains to be established. The authors express their thanks to Ralph Hill and Gwen Moriarty. The report‘is based in part on work performed under contract with the Atomic Energy Commission at the University of Rochester Atomic Energy Project ExptI Cd Res 63
and assigned Report Number UR-1192 and in part on work supported by USPH Research Grants CA03589 and 5ROl-AM08271. A. R. Terepka is a USPHS Career Development Awardee.
REFERENCES Terepka, A R, Stewart, M E & Merkel, N, Exptl cell res 58 (1969) 107. Johnson, P M & Comar, C L, Am j physiol 183 (1955) 365. Stewart, M & Terepka, A R, Exptl cell res 58 (1969) 93. Carroll, K G & Tullis, J L, Nature 217 (1968) 1172 Received July 9, 1970
Aggregation of dissociated mouse blastomeres T. P. LIN and JOAN FLORENCE, Department of Anatomy, School of Medicine, University of California, San Francisco, Calif. 94122, USA
Dissociated blastomeres of four- or eight-cell mouse eggs from the same or different strains can reaggregate and some develop to blastocyst stage in droplet cultures.
Studies on the dissociation and aggregation of embryonic cells are helpful in elucidating the mechanism of cellular interaction in multicellular organization. Dissociated cells from various embryonic tissues or organ rudiments can be maintained in vitro during which time they can aggregate and establish tissue-like structures [7]. Studies on cell interaction in mammalian development have been performed by fusion of entire early embryos [6, 91 or by injection of blastomeres into the cavity of blastocysts [4]; in such casesthe fused or added cells interacted with those in the embryos and all retained their capacity of regulation. Since interaction of dissociated young embryonic cells of sea urchins has resulted in restitution and development to pluteus-like larvae [5], it seemed possible that the dissociated blastomeres of early mouse embryos might be re-
J. R. Coleman et al.
permit the complete separation of chromosomes of one specific size from other chromosomes. Perhaps this difficulty could be resolved by using cell types in which there is a real diversity in chromosome size. The spectrum of morphological diversity among the 44 chromosomes of the Syrian hamster complement is greater than that in either the HeLa cell or L cell complements. However, even the fractionation of these chromosomesdid not result in a greatly improved separation of individual types of chromosomes from one another. Better success might be obtained by using cell types such as those from Microtus agrestis, in which there is an extreme size difference between the X chromosomes and the autosomes. This work forms part of a doctoral dissertation submitted by G. D. Burkholder to the Faculty of Graduate Studies and Research, McGill University. The investigation was supported by Grant MT2169 from the Medical Research Council of Canada, and a postgraduate scholarship from the National Research Council of Canada.
REFERENCES 1. Burkholder, G D & Mukherjee, B B, Exptl cell res 61 (1970) 413. 2. Huberman, J A & Attardi, G, J cell biol 31 (1966) 95. 3. 1 J mol biol 29 (1967) 487. 4. Maio, J J & Schildkraut. C L. J mol biol 24 (1967) 29. 5. - Ibid 40 (1969) 203. 6. Matsuya, Y & Yamane, I, Exptl cell res 50 (1968) 652. 7. Mendelsohn, J, Moore, D E & Salzman, N P, J mol biol 32 (1968) 101. 8. Robbins, E & Marcus, P I, Science 144 (1964) 1152. 9. Salzman, N P, Moore, D E & Mendelsohn, J, Proc natl acad sci US 56 (1966) 1449. 10. Schneider, E L & Salzman, N P, Science 167 (1970) 1141. 11. Terasima, T & Tolmach, L J, Exptl cell res 30 (1963) 344. Received June 25, 1970
Exptl Cell Res 63
Electron probe analysis of calcium distribution during active transport in chick chorioallantoic membrane J. R. COLEMAN, S. M. DeWITT, P. BATT and A. R. TEREPKA, Department of Radiation Biology and Biophysics, University of Rochester, School of Medicine and Dentistry, Rochester, N. Y. 14620, USA Summary The embryonic chick chorioallantoic membrane actively transports calcium and the transported calcium is compartmentalized or sequestered, since it does not mix completely with calcium already contained within the membrane. Correlated electron probe and electron microscope analysis shows that only certain ectodermal cells of the membrane contain concentrations of calcium. We suggest that these are specialized cells which are specifically involved in trans-epithelial transport of calcium and represent containing the sequestered the “compartments” calcium.
The ectoderm of the embryonic chick chorioallantoic membrane (CAM) develops the ability to transport calcium against an electrochemical gradient after about 13-14 days of egg incubation [l], a time which corresponds with the rapid movement of calcium from the eggshell to the embryo for skeletal calcification [2]. Transport chamber studies of the isolated membrane using 45Casuggestedthat the calcium being transported through the membrane was compartmentalized [ 11.Even after 6 h of exposure to 45Ca,the spec. act. of the membrane was only about twothirds that of the outside bathing solution. However, the spec.act. of the calcium transported to the inside solution was significantly higher than that of the membrane and close to that of the outside solution from which the calcium was transported. The ectoderm of the CAM is an epithelium forming a continuous layer of cells under the non-cellular egg shell membranes. It is composed of at least three different cell types supporting and surrounding respiratory capillaries [3]. The “compartment” sequestering calcium in transit could be restricted to a
Calcium in chorioallantoic membrane
certain type of cell in the epithelium or could be a separate compartment within every cell. To help distinguish between these two alternatives, we used the electron probe X-ray microanalyzer to localize sites of calcium concentration within the CAM. If the distribution of calcium found in the CAM were homogeneous, it would indicate that each cell contains a “compartment” for the calcium in transport. The electron probe revealed, however, that the calcium was concentrated only within certain cells, suggesting that these specialized epithelial cells were responsible for the active calcium transport by this membrane. Material and Methods Tissues were fixed for 30 min to 1 h with 6% acrolein (synthesized by Dr W. G. Aldridge, University of Rochester) in 0.1 M cacodylate buffer (pH 7.2) containing 1% sodium oxalate. This was followed by a short wash in the buffer, a 30 min postfixation with 1% 0~0, in the same buffer, dehydration in ethanol and embedding in Araldite@ or paraffin. Preliminary studies had shown that this procedure produced no substantial loss of total calcium or ““Ca and provided good electron microscope morphology. Two-micron thick sections of CAM were cut without exposure to water, mounted on silicon wafers [5] dry or after expansion on 5 % sodium oxalate at 60°C and coated lightly with vacuumevaporated aluminium or carbon after removal of paraffin with xylene or benzene. These were examined with an Applied Research Laboratory Electron Probe X-ray M&oanalyzer, EMX, usually at 22 kV accelerating potential and about 7 x lo-+’ A sample current, with a beam less than 1 pm in diameter. Images were recorded from the oscilloscope screen on Polaroid@ film. The CAM was removed from the incubated egg and mounted in an Ussing-type transport chamber. Oxygenated Krebs-Ringer bicarbonate solution containing 1.0 mM calcium was circulated on both sides and 46Ca was added to the outside (ectoderm) solution. Samples from the inside and outside solutions were taken at intervals and assayed for total calcium and 46Ca to insure that the CAM was actively transporting calcium [l]. The membrane was then removed from the chamber, fixed, dehydrated, embedded, sectioned and examined with the probe as described above. Adjacent, or nearly adjacent, thin sections of Araldite@ embedded tissues were sectioned and prepared in a conventional manner for electron microscopy. Specimens of CAM were prepared from eggs incubated for at least 14 days and known to be actively transporting calcium, and from eggs incubated less
217
than 13 days so that the active transport process had not started. Also examined were- membranes in which calcium transport was inhibited by incubation in a nitrogen atmosphere and mature-membranes exposed to a solution containing strontium which is also actively transported but at a rate about onethird that of calcium [l].
Results
Electron probe analysis showed that discrete calcium localizations occurred only in the ectodermal cell layer in mature, transporting CAM (fig. 1a). The corresponding sample current image of this tissue section is shown in fig. 1 b. Phosphorus and sulfur scans of the specimen (fig. 1 c, d) showed generalized distributions which agree with the known chemical concentrations of these elements. The calcium concentrations visualized with the electron probe appeared to be limited to certain ectodermal cells which were found immediately adjacent to respiratory capillaries in the corresponding sample-current images (fig. 2a, b). These cells could be identified in the electron microscope as having long cytoplasmic processes extending between the endothelial cells of capillaries and the shell membrane (fig. 2 c). The cytoplasmic processesthat lay between the capillaries and the shell membrane contain 400 to 1 000 A diameter vesicles. The mitochondria of these cells cluster around the nucleus which is usually located near the basal surface and some distance from the calciumcontaining cytoplasmic processes adjacent to the blood capillaries. No discrete calcium localizations were found with the electron probe in young, non-transporting membranes or in membranes that had been incubated in nitrogen, even though both of these membranes had been exposed to the same concentration of calcium as the mature, transporting membranes. When a mature, transporting membrane was exposed to strontium rather than calcium, strontium localizations were found at Exptl Cell Res 63
218
J. R. Coleman et al.
Fig. 1. u-d are X-ray and sample current images of the same section of chorioallantoic membrane (CAM) in the electron probe at a magnification of 10 ,um per screen division. The X-ray images are the result of long photographic integrations (40 min). Signals not associated with tissue components and randomly distributed through the background are due to noise and cannot be ascribed to the presence of any particular element. (a) Calcium X-ray image of CAM section showing calcium restricted to discrete sites within the membrane. The exact location of these can be determined by referring to the grid coordinates on the sample current image in 1b. (6) Sample current image showing shell membrane (SM) above the respiratory capillaries which appear as dark areas in the ectodermal cell layer (EC). A large blood vessel (BP) containing red blood cells is in the mesoderm (M) and the thin endodermal layer (EN) is seen near the bottom of the picture. Three sites of calcium localization are marked by arrows; the others can be determined from coordinates of grid lines in the calcium X-ray image. (c) Sulfur X-ray image of same section showing concentration of sulfur mainly restricted to noncellular shell membrane (SM). Note that sulfur signal is not restricted to discrete sites as is calcium. (6) Phosphorus X-ray image of same section showing concentration of phosphorus reflecting distribution of cellular material throughout the CAM.
the same sites as calcium. Since membranes not exposed to strontium experimentally had no detectable strontium by probe analysis, this indicates that the observed concentration sites contained the divalent ions which were being actively transported by the membrane. The validity of these electron probe analyses, as with all histochemical techniques, is dependent upon tissue preparation proceExptl Cell Res 63
dures that neither lose nor redistribute calcium and yet preserve cell morphology. In the studies reported here, calcium loss was assessedby incubating mature, transporting membranes with 45Caas described [I], then dividing the membrane in two. One-half was analyzed directly for total calcium and 45Ca and the second half was analyzed after being subjected to the fixing and embedding proce-
Calcium in chorioallantoic membrane 219
Fig. 2. a and b are, respectively, sample current image and the corresponding calcium X-ray image of the same section at a magnification of 5 pm/grid square. In (a) portions of the shell membrane (S&f), ectoderm (E) and mesoderm (M) are visible. Directly beneath the shell membrane: ectoderm junction are capillaries which appear as dark spaces and contain three bright, roughly disc-shaped erythrocytes, one of which is labelled R. Between the two capillaries is an ectodermal cell (similar to that seen between two capillaries in (c) which extends from the interior of the ecoderm to the shell membrane and lies between the endothelial lining of the capillaries and the shell membrane. In (b) only a small discrete region gives evidence of calcium signals above background levels. This corresponds to a portion of the ectodermal cell seen between the two capillaries in (A) and is restricted to the region of the cell near the shell membrane. (c) Electron micrograph of thin section of CAM in a region similar to that seen in (a) showing the type of ectodermal cell seen between two capillaries. The ectodermal cell has long thin cytoplasmic processes that extend between the noncellular shell membrane (SW) and the endothelial cells of the capillaries (~5).These ectodermal cells are the sites of calcium concentration as determined by electron probe analysis. (Bar represents 1 ym.)
Exptl Cell Res 63
220
T. P. Lin & Joan Florence
dures. Total calcium content as well as the spec. act, of both halves were comparable. While no independent method to assess redistribution exists, we took the following indirect evidence to suggest that redistribution, if it occurred, was minimal, and was beyond the resolution of the electron probe: (1) When conditions which were likely to cause redistribution (e.g. distortion and destruction of cell morphology, floating sections on water) were employed, only a generalized distribution of calcium was observed. (2) Calcium localizations were found only in mature, transporting membranes. Significantly, these were not randomly distributed but were associatedwith a particular cell type occupying a characteristic site within the ectoderm. Electron microscope examination of these cells gave no indication of crystalline deposits. (3) Calcium chloride ranging x 100 above and below the amount found in membranes was dissolved in 10% albumen which was then processedby the methods indicated. No discrete localizations were found and calcium remained homogeneously distributed. In conclusion, we suggestthat the compartment which sequesterscalcium and prevents it from mixing with calcium already within the membrane is restricted to certain ectodermal cells which are specifically involved in active calcium transport. These cells surround the respiratory capillaries with long cytoplasmic processes. Such a juxtaposition would facilitate the movement of eggshell calcium into the embryo’s blood plasma. Whether the vesicles noted in the cells that contain calcium localizations are directly involved with the transport of calcium [l] remains to be established. The authors express their thanks to Ralph Hill and Gwen Moriarty. The report‘is based in part on work performed under contract with the Atomic Energy Commission at the University of Rochester Atomic Energy Project ExptI Cd Res 63
and assigned Report Number UR-1192 and in part on work supported by USPH Research Grants CA03589 and 5ROl-AM08271. A. R. Terepka is a USPHS Career Development Awardee.
REFERENCES Terepka, A R, Stewart, M E & Merkel, N, Exptl cell res 58 (1969) 107. Johnson, P M & Comar, C L, Am j physiol 183 (1955) 365. Stewart, M & Terepka, A R, Exptl cell res 58 (1969) 93. Carroll, K G & Tullis, J L, Nature 217 (1968) 1172 Received July 9, 1970
Aggregation of dissociated mouse blastomeres T. P. LIN and JOAN FLORENCE, Department of Anatomy, School of Medicine, University of California, San Francisco, Calif. 94122, USA
Dissociated blastomeres of four- or eight-cell mouse eggs from the same or different strains can reaggregate and some develop to blastocyst stage in droplet cultures.
Studies on the dissociation and aggregation of embryonic cells are helpful in elucidating the mechanism of cellular interaction in multicellular organization. Dissociated cells from various embryonic tissues or organ rudiments can be maintained in vitro during which time they can aggregate and establish tissue-like structures [7]. Studies on cell interaction in mammalian development have been performed by fusion of entire early embryos [6, 91 or by injection of blastomeres into the cavity of blastocysts [4]; in such casesthe fused or added cells interacted with those in the embryos and all retained their capacity of regulation. Since interaction of dissociated young embryonic cells of sea urchins has resulted in restitution and development to pluteus-like larvae [5], it seemed possible that the dissociated blastomeres of early mouse embryos might be re-