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Some observations on the surface coat and intercellular matrix material of the amphibian ectoderm
378
Experimental
Cell
Research
20, 378-383
(I 960)
SOME OBSERVATIONS ON THE SURFACE COAT AND INTERCELLULAR MATRIX MATERIAL OF THE AMPHIBIAN ECTODERM’ E. BELL Department
of Biology,
Massachusetts
Institute
Received
August
of Technology,
Cambridge,
Mass.,
U.S.A.
12, 1959
HOLTFRETER [6] described the surface coat in Rana pipiens and suggested that it was made up primarily of protein. More recently, Brachet [4] observed that the intercellular matrix of the amphibian ectoderm (which appears to be continuous with the surface coat) is an RNA-protein calcium complex. It was previously reported [2, 31 that the surface coat could be removed from Rana pipiens embryos by means of focused ultrasound. The technique provided a method for isolation of the surface coat which could then be studied apart from the embryo. The present paper is a summary of preliminary cytochemical and electron microscopic observations on the surface coat. It will be shown that the coat and the intercellular matrix material of the ectoderm are similar cytochemically and consist primarily of mucopolysaccharide. In the surface coat are embedded many tine granules; it undergoes a process of morphogenesis, being initially structureless, but later gives evidence of containing a disorganized array of osmotically sensitive tibrils. METHODS
AND
MATERIALS
The experimental procedure for irradiating embryos and larvae with focused ultrasound has been described elsewhere [3]. Prior to irradiation, embryos or larvae are incubated in PVP and distilled water 1: 1 for 90 minutes, then washed three times in 20 per cent Holtfreter’s solution and exposed for 1 to 3 secondsto ultrasound of 4 megacycles frequency and 260 w/cm2 peak intensity. Exposure to ultrasound results in the elevation from the entire embryo of what appeared to be a thin, filmy membrane (Fig. 1). Up to Stage 14 the membrane could be removed without cells, whereas after Stage 14 a small dispersepopulation of ectodermal cells which represented about 1 to 2 per cent of the total number of surface cellsof the germ were found embedded in the membrane or surface coat upon its removal from the embryo. For cytochemical or electron microscopic examination the surface coat was spread on to a glass slide or onto a filmed or unfilmed grid while still in Holtfreter’s solution. 1 This investigation was supported by a research of Health, United States Public Health Service. Experimenf
al Cell Research
20
grant
(B1257-C2)
from
the National
Institutes
The amphibian
surface coat
379
RESULTS
At Stage 17, the surface coat appeared to he intimately associated with the membranes, and possibly the cytoplasm, of the peridermal cells. These cells mere, in some instances, nearly completely enveloped by coat material. The impression was distinctly given that the coat not only covered the surface of the germ but also dipped down between the cells of the periderm to join with or become the intercellular matrix (Fig. 2). By none of the staining procedures employed was it possible to distinguish between the surface coat and the intercellular matrix material with which it was continuous. Unna’s methyl green pyronin stain was used to determine whether RSA was present in the coat. A very strong positive reaction was observed in the material at all stages but it was not possible to abolish the reaction by ribonuclease digestion. This would indicate that the reaction is not specific for Rh’A but that certain embryonic mucopolysaccharides react positively to pyronin. It is tentatively suggested, until other tests are made, that little or no RNA is present in the membrane. Retween Stages 13 and 23, or late yolk plug and half-grown tadpole, the coat and matrix are strongly PAS positive. The reaction increases in intensity between Stages 13 and 23. Saliva has no effect upon the intensity of staining. Coat and matrix material from Stage 13 to 23, embryos and larvae give a strong metachromatic reaction with toluidine blue. The material was stained with either a 0.1 per cent solution of toluidine blue for 5 minutes or with a 5 per cent solution of toluidine blue for 20 minutes. The latter gave a more pronounced metachromatic reaction. Digestion with hyaluronidase abolished the reaction completely and showed that the coat and matrix \vere rich in mucopolysaccharide. It hvas not possible to demonstrate the presence of protein by the use of the ninhydrin, hlillon, or the biuret reagents. -4 modification of the nitroprusside test, more sensitive than the previous three, also failed to give a positive reaction. The former tests were carried out on coat material from embryos or larvae between Stages 13 and 23. Coat and matrix did react positively to bromophenol blue which indicated the presence of basic amino acids; the negative nitroprusside reaction indicates that it probably lacks sulphur-containing amino acids. The surface coat of the Stage 13 or 14 embryo had little or no structure when viewed under the electron microscope. One could see only the very distinct pigment granules against an amorphous background. Holtfreter’s suggestion that the surface coat contains yolk particles has not been confirmed. 2.5 - 60173253
Experimental
Cell
Research
20
380
Fig. l.-Portion Coat is transparent
E. Bell
of uutrcatetl and cannot
surfarc coat from Stage 19 larva be tlistingnishcd from background.
with
attached
pcritlermal
cells.
Lit Stage 17 or 18, the first signs of structure \\-ere observecl in the coat and matrix material which, at this time, contained tiny coiled fibrils. 13~ Stage 19, the fibrils appeared to he more numerous and \\-ere about 0.06 i4 in diameter and two to three microns long (Fig. 3). The fibrils \vere found to he very fragile and seemed to he sensitivta to changes in osmotic pressure. \Yhen membranes were stretched, unfixed, over a grid anti allowed to dry, leashing I\-ith distilled \vatcr resulted in the disappearance of the fihrils. If the material Al-as fixed in osmium tetrositie dichromatc or phosphotungstic acid, the fihrils disappeared and the coat collapsed. \\‘ashing with Holtf’reter’s solution did not atTect them. ITntreatcd coats stretched oyer an unfilmed grid held up even under a strong electron heam. Fig. 2.pSurface coat and intercellular matrix material blue. The coat and matrix gave a strong metachromatic pretreatment with hgaluronidasc.
from
Stage reaction
larva stain4 with toluidinc whirh could br aholishetl 1))
15
Fig. 3.-Electron micrograph of fibrils in surface coat and intercellular matrix larva. Note terminal dense regions and fibrillae of small caliber (0.01 ,U tliamctcr) to make up the fibril (O.O6 p diameter). .\Iagnification 17.000
from Stag? 19 which appear
The amphibian
surface coat
381
382
E. Bell
At a magnification of 38,000 some details of the fine structure of the fihril were apparent. The fibril appeared to consist of laminae or tubules. Terminal electron dense regions were observed on both ends of each fibril and there is some evidence that the fibril can break down into librillae of smaller caliber (Fig. 3) which measure about 0.01 p in diameter. DISCUSSION
The surface coat appears to be indistinguishable cytochemically and structurally from the intercellular matrix material of the ectoderm or epidermis. The coat can be conceived of initially as an envelope which is closely applied to the plasma membrane of the egg. It seems probable that as cleavage occurs, coat material is carried inward toward the blastocoel and that ultimately epidermal cells are surrounded at least in part by some material of coat origin. This is in accord with the observation of Schechtman [lo], Nicholas [8], Nieuwkoop [9], and Ballard [ 11, all of whom have reported cortical ingression during cleavage. The coat and matrix are primarily mucopolysaccharide in character and might therefore provide the kind of substrate which is necessary for the extensive cell movements that occur on the surface of the germ during development. Although there is no evidence that the matrix confers mobility on the cells of the ectoderm, this possibility is suggested. It has been proposed by Meier [7] that polysaccharides may be instrumental in stimulating otherwise quiescent cells to move. The appearance of fibrils in the dried coat and matrix material at about Stage 18 may explain the fact recently reported by Curtis [5] that dissociation of Xenopus ectoderm by EDTA no longer occurs soon after neural tube closure. It is possible that the fibril may serve as a reinforcement of the already existing bonds between cells of the ectoderm. This suggestion was forecast by Weiss [ll]. It is curious, however, that fibrils occur in a material apparently so poor in protein. SUMMARY
An intercellular membrane-like matrix has been isolated from the ectoderm of Rana pipiens embryos and larvae. Initially structureless, by Stage 18 it becomes filled with osmotically sensitive fibrils. The surface coat or intercellular substance appears to be largely mucopolysaccharide in character and probably contains basic amino acids, but no sulphur-containing amino acids. The surface coat and intercellular matris seem cytochemicallp and structurally indistinguishable. Experimental
Cell Research
20
The amphibian
surface coat
The technical assistance of Mrs. L. M. Fessenden and Mr. E. M. Kaighn fully acknowledged.
is grate-
REFERENCES
1. BALLARD, W. W., J. Exptl. 2001. 129, 77 (1955). 2. BELL, E., Anat. Record 131, 532 (1958). 3. ___ in Proceedings of the First National Biophysics Conference, Yale University Press, New Haven, pp. 674-682, 1959. 4. BRACIIET, J., Biochemical Cytology. Academic Press, Sew York, pp. 410-11, 1957. 5. CURTIS, A. S., Proc. Roy. Phys. Sot. Edinburgh 26, 25 (1957). 6. HOLTFRETER, J., J. Exptl. Zool. 93, 251 (1943). 7. MEIER, R., Chemistry and Biology of Mucopolysaccharitles. Little, Brown, and Company, Boston, 1958. 8. NICHOLAS, S. J.. J. Exptl. Zool. 100, 265 (1945). 9. h'IEUWKO&', P.‘D., Ar;h. NCerl. Zok. 8, i (1947). 10. SCHECHTMAK, A. M., Univ Calif. Publ. Viol. Sci. 39, 303 (1934). 11. WEISS, P., Quart. Rev. Rio!. 25, 177 (1950).
Experimental
Cell
Research
20
Experimental
Cell
Research
20, 378-383
(I 960)
SOME OBSERVATIONS ON THE SURFACE COAT AND INTERCELLULAR MATRIX MATERIAL OF THE AMPHIBIAN ECTODERM’ E. BELL Department
of Biology,
Massachusetts
Institute
Received
August
of Technology,
Cambridge,
Mass.,
U.S.A.
12, 1959
HOLTFRETER [6] described the surface coat in Rana pipiens and suggested that it was made up primarily of protein. More recently, Brachet [4] observed that the intercellular matrix of the amphibian ectoderm (which appears to be continuous with the surface coat) is an RNA-protein calcium complex. It was previously reported [2, 31 that the surface coat could be removed from Rana pipiens embryos by means of focused ultrasound. The technique provided a method for isolation of the surface coat which could then be studied apart from the embryo. The present paper is a summary of preliminary cytochemical and electron microscopic observations on the surface coat. It will be shown that the coat and the intercellular matrix material of the ectoderm are similar cytochemically and consist primarily of mucopolysaccharide. In the surface coat are embedded many tine granules; it undergoes a process of morphogenesis, being initially structureless, but later gives evidence of containing a disorganized array of osmotically sensitive tibrils. METHODS
AND
MATERIALS
The experimental procedure for irradiating embryos and larvae with focused ultrasound has been described elsewhere [3]. Prior to irradiation, embryos or larvae are incubated in PVP and distilled water 1: 1 for 90 minutes, then washed three times in 20 per cent Holtfreter’s solution and exposed for 1 to 3 secondsto ultrasound of 4 megacycles frequency and 260 w/cm2 peak intensity. Exposure to ultrasound results in the elevation from the entire embryo of what appeared to be a thin, filmy membrane (Fig. 1). Up to Stage 14 the membrane could be removed without cells, whereas after Stage 14 a small dispersepopulation of ectodermal cells which represented about 1 to 2 per cent of the total number of surface cellsof the germ were found embedded in the membrane or surface coat upon its removal from the embryo. For cytochemical or electron microscopic examination the surface coat was spread on to a glass slide or onto a filmed or unfilmed grid while still in Holtfreter’s solution. 1 This investigation was supported by a research of Health, United States Public Health Service. Experimenf
al Cell Research
20
grant
(B1257-C2)
from
the National
Institutes
The amphibian
surface coat
379
RESULTS
At Stage 17, the surface coat appeared to he intimately associated with the membranes, and possibly the cytoplasm, of the peridermal cells. These cells mere, in some instances, nearly completely enveloped by coat material. The impression was distinctly given that the coat not only covered the surface of the germ but also dipped down between the cells of the periderm to join with or become the intercellular matrix (Fig. 2). By none of the staining procedures employed was it possible to distinguish between the surface coat and the intercellular matrix material with which it was continuous. Unna’s methyl green pyronin stain was used to determine whether RSA was present in the coat. A very strong positive reaction was observed in the material at all stages but it was not possible to abolish the reaction by ribonuclease digestion. This would indicate that the reaction is not specific for Rh’A but that certain embryonic mucopolysaccharides react positively to pyronin. It is tentatively suggested, until other tests are made, that little or no RNA is present in the membrane. Retween Stages 13 and 23, or late yolk plug and half-grown tadpole, the coat and matrix are strongly PAS positive. The reaction increases in intensity between Stages 13 and 23. Saliva has no effect upon the intensity of staining. Coat and matrix material from Stage 13 to 23, embryos and larvae give a strong metachromatic reaction with toluidine blue. The material was stained with either a 0.1 per cent solution of toluidine blue for 5 minutes or with a 5 per cent solution of toluidine blue for 20 minutes. The latter gave a more pronounced metachromatic reaction. Digestion with hyaluronidase abolished the reaction completely and showed that the coat and matrix \vere rich in mucopolysaccharide. It hvas not possible to demonstrate the presence of protein by the use of the ninhydrin, hlillon, or the biuret reagents. -4 modification of the nitroprusside test, more sensitive than the previous three, also failed to give a positive reaction. The former tests were carried out on coat material from embryos or larvae between Stages 13 and 23. Coat and matrix did react positively to bromophenol blue which indicated the presence of basic amino acids; the negative nitroprusside reaction indicates that it probably lacks sulphur-containing amino acids. The surface coat of the Stage 13 or 14 embryo had little or no structure when viewed under the electron microscope. One could see only the very distinct pigment granules against an amorphous background. Holtfreter’s suggestion that the surface coat contains yolk particles has not been confirmed. 2.5 - 60173253
Experimental
Cell
Research
20
380
Fig. l.-Portion Coat is transparent
E. Bell
of uutrcatetl and cannot
surfarc coat from Stage 19 larva be tlistingnishcd from background.
with
attached
pcritlermal
cells.
Lit Stage 17 or 18, the first signs of structure \\-ere observecl in the coat and matrix material which, at this time, contained tiny coiled fibrils. 13~ Stage 19, the fibrils appeared to he more numerous and \\-ere about 0.06 i4 in diameter and two to three microns long (Fig. 3). The fibrils \vere found to he very fragile and seemed to he sensitivta to changes in osmotic pressure. \Yhen membranes were stretched, unfixed, over a grid anti allowed to dry, leashing I\-ith distilled \vatcr resulted in the disappearance of the fihrils. If the material Al-as fixed in osmium tetrositie dichromatc or phosphotungstic acid, the fihrils disappeared and the coat collapsed. \\‘ashing with Holtf’reter’s solution did not atTect them. ITntreatcd coats stretched oyer an unfilmed grid held up even under a strong electron heam. Fig. 2.pSurface coat and intercellular matrix material blue. The coat and matrix gave a strong metachromatic pretreatment with hgaluronidasc.
from
Stage reaction
larva stain4 with toluidinc whirh could br aholishetl 1))
15
Fig. 3.-Electron micrograph of fibrils in surface coat and intercellular matrix larva. Note terminal dense regions and fibrillae of small caliber (0.01 ,U tliamctcr) to make up the fibril (O.O6 p diameter). .\Iagnification 17.000
from Stag? 19 which appear
The amphibian
surface coat
381
382
E. Bell
At a magnification of 38,000 some details of the fine structure of the fihril were apparent. The fibril appeared to consist of laminae or tubules. Terminal electron dense regions were observed on both ends of each fibril and there is some evidence that the fibril can break down into librillae of smaller caliber (Fig. 3) which measure about 0.01 p in diameter. DISCUSSION
The surface coat appears to be indistinguishable cytochemically and structurally from the intercellular matrix material of the ectoderm or epidermis. The coat can be conceived of initially as an envelope which is closely applied to the plasma membrane of the egg. It seems probable that as cleavage occurs, coat material is carried inward toward the blastocoel and that ultimately epidermal cells are surrounded at least in part by some material of coat origin. This is in accord with the observation of Schechtman [lo], Nicholas [8], Nieuwkoop [9], and Ballard [ 11, all of whom have reported cortical ingression during cleavage. The coat and matrix are primarily mucopolysaccharide in character and might therefore provide the kind of substrate which is necessary for the extensive cell movements that occur on the surface of the germ during development. Although there is no evidence that the matrix confers mobility on the cells of the ectoderm, this possibility is suggested. It has been proposed by Meier [7] that polysaccharides may be instrumental in stimulating otherwise quiescent cells to move. The appearance of fibrils in the dried coat and matrix material at about Stage 18 may explain the fact recently reported by Curtis [5] that dissociation of Xenopus ectoderm by EDTA no longer occurs soon after neural tube closure. It is possible that the fibril may serve as a reinforcement of the already existing bonds between cells of the ectoderm. This suggestion was forecast by Weiss [ll]. It is curious, however, that fibrils occur in a material apparently so poor in protein. SUMMARY
An intercellular membrane-like matrix has been isolated from the ectoderm of Rana pipiens embryos and larvae. Initially structureless, by Stage 18 it becomes filled with osmotically sensitive fibrils. The surface coat or intercellular substance appears to be largely mucopolysaccharide in character and probably contains basic amino acids, but no sulphur-containing amino acids. The surface coat and intercellular matris seem cytochemicallp and structurally indistinguishable. Experimental
Cell Research
20
The amphibian
surface coat
The technical assistance of Mrs. L. M. Fessenden and Mr. E. M. Kaighn fully acknowledged.
is grate-
REFERENCES
1. BALLARD, W. W., J. Exptl. 2001. 129, 77 (1955). 2. BELL, E., Anat. Record 131, 532 (1958). 3. ___ in Proceedings of the First National Biophysics Conference, Yale University Press, New Haven, pp. 674-682, 1959. 4. BRACIIET, J., Biochemical Cytology. Academic Press, Sew York, pp. 410-11, 1957. 5. CURTIS, A. S., Proc. Roy. Phys. Sot. Edinburgh 26, 25 (1957). 6. HOLTFRETER, J., J. Exptl. Zool. 93, 251 (1943). 7. MEIER, R., Chemistry and Biology of Mucopolysaccharitles. Little, Brown, and Company, Boston, 1958. 8. NICHOLAS, S. J.. J. Exptl. Zool. 100, 265 (1945). 9. h'IEUWKO&', P.‘D., Ar;h. NCerl. Zok. 8, i (1947). 10. SCHECHTMAK, A. M., Univ Calif. Publ. Viol. Sci. 39, 303 (1934). 11. WEISS, P., Quart. Rev. Rio!. 25, 177 (1950).
Experimental
Cell
Research
20