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Localization of lens antigens in developing frog embryos
Experimental
Cell Research
21, 541-547
LOCALIZATION
541
(1960)
OF LENS
ANTIGENS
FROG
of Zoology,
State
Received
DEVELOPING
EMBRYOS
R. A. FLICKINGER Department
IN
and G. STONE University
December
of Iowa,
Zowa,
U.S.A.
12, 1959
1~ is of some interest to learn if proteins with organ specific combining groups are present in the embryo prior to the differentiation of the organ. The lens is a particularly good organ to use in a serological investigation in relation to embryonic development [la] since it has some degree of organ specificity and there is information from biological experiments concerning the time period when certain tissues can form lenses [‘i, 8, 9, 101.
MATERIALS
AND
METHODS
Antisera were obtained to centrifugal supernates (10,000 xg) of homogenates of frog lenses (Rana pipiens), cattle lenses, immature frog ovaries and frog testes. The homogenates used in preparing injection and test antigens were prepared with an equal volume of tissue and 0.9 per cent NaCl containing merthiolate at a l/10,000 concentration. Protein concentrations of the test antigen preparations were ascertained by nitrogen determinations (Nesslerization) and the use of the factor 6.25. The antisera were produced by injections given every other day to young 6-8 lb. New Zealand rabbits. The injection schedule consisted of intravenous injections of 0.5, 1.0, 2.0, 3.0 and 1.0 ml with a 3.0 ml intraperitoneal injection given with the last intravenous injection. Recall injecstions, consisting of a 1.0 ml intravenous and a 3.0 ml intraperitoneal injection, were given three weeks after the completion of the first injection series. The animals were bled 6 days after the recall injections. Ouchterlony agar plate diffusion experiments were carried out according to the methods of Bjiirklund [l]. RESULTS Precipitin reactions.-The frog lens antiserum was not specific and also reacted against serum and other organs, while the anti-beef lens serum shovved a weak cross reaction with a frog brain supernatc. However, after absorption of one volume of the anti-frog lens serum with 24 volumes of 1 This investigation P’oundation.
was
supported
by
a research
grant
(6139)
from
the
Experimental
National
Science
Cell Research
21
542
R. A. Flickinger
and G. Stone
brain supcrnate in four absorptions, and similar absorption of the anticattle lens serum, these antisera did not react against centrifugal supernates of homogenates prepared from brain, kidney, liver, heart, muscle, stomach, skin and testes; nor did they react with frog serum. The absorbed anti-frog lens serum reacted with a l/SO,000 dilution of the frog lens antigen, which contained 1.25 ,~ugprotein/ml. The unahsorhed anticattle lens serum reacted w?th a 1 /GO,000 dilution of the frog lens antigen, xvhich contained 1.6 ,~ug protein/ml. Roth the anti-frog and anti-cattle lens sera reacted against a l/l0 dilution of frog lens antigen at an antiserum dilution of at least l/10,000. 13oth the frog and the cattle lens antisera, after having been absorbed hvith the brain supernate, reacted positively with the centrifugal supernate of the immature oocytes (small oocyte supernate) at a l/20 dilution. This contained 560 pug protein/ml. Dilution of the two antisera to a 1 jl0 concentration aholished activity. It is known that lens determination occurs hy the neurula stage in Runn esczrlenfa embryos raised at 10-l ‘L”C, hut at 25°C the optic vesicle must play a role in lens determination [l 11. In our experiments two groups of Rnnrr pipiens embryos \\-cre raised to the late neurula stage (Shurn\\-ag stage 15) at two difrerent temperatures (10’ and 25’C). Thcsc embryos \vcre surgically separated into head and trunk regions and centrifugal supernatcs QY’~C’ reacted against the anti-lens sera in precipitin reactions. The head and trunk fractions of the embryos raised at 10’ and ‘25°C reacted equally xvell with absorbed anti-frog lens serum, but did not react with anti-cattle lens strum. Positive reactions were obtained with l/l0 (hut not l/20) antigen dilutions of the 10” and 25°C head fractions which contained approximately 2,000 ,LL~ protein/ml. The trunk antigens (10’ and 25°C) did not react at a l/l0 antigen dilution; ho\vcvcr, their protein concentration was about half that of the head fractions. The prospective otic capsule, optic vesicle and oral sucker areas were’ one hundred early tailbud Rnnrt pipiens extirpated from approximately embryos at Shumway stage 16, prior to any morphological specialization of these structures. The centrifugal supcrnatcs from these areas were utilized as test antigen preparations and each reacted within t\\-enty minutes in precipitin reactions with anti-frog lens serum that had been partially absorbed three times with brain supernate. They were inactive against the anti-frog serum that had been completely absorbed by four brain supernate absorptions. This inactivity is ascribed to further dilution of the antiserum. The tails of late stage feeding larvae of Rnnrr pipiens (A-80 mm) were Experimental
Cell Research
21
Lens antigen
localization
prepared anti-frog
as centrifugal supernates and found to react with the brain absorbed antigen dilution of Rana and anti-cattle lens sera. The limiting pipiens larval tail fraction which reacted with the brain absorbed anti-frog lens serum was l/10. This contained 212 pg protein/cc. The brain absorbed anti-frog lens serum gave a positive reaction at a l/l0 dilution of antiserum. A ten fold dilution of anti-cattle lens serum was inactive. The aqueous humor and the iris-pigmented retina were removed from the eyes of adult frogs (Rana pipiens) and from two species of salamanders (Triturus (Taricha) torosus and T. rivularis). The anti-frog lens serum reacted with a l/25 dilution of frog aqueous humor (51 pg protein/ml) and a l/l0 dilution of salamander aqueous humor. The frog lens antiserum was still active against undiluted frog aqueous humor (275 ,ug protein/ml) at a l/l0 antiserum dilution. Only the undiluted anti-cattle lens serum reacted with frog aqueous humor. The undiluted iris-pigmented retina centrifugal supernate (1,250 lug protein/ml) reacted with the anti-frog lens and anti-cattle lens sera. A l/l0 dilution of frog lens antiserum could still react with the irisretina test antigen. Agar plate diffusion reactions.-In order to determine if the antigens in the immature oocytes, brain, serum, whole feeding larvae and the larval tail were similar or dissimilar to those in the aqueous humor, retina and the lens itself, unabsorbed anti-frog lens serum was utilized in a number of agar plate diffusion tests. The results are shown in Figs. 1 and 2. These drawings represent a combination of the results of a large series of Ouchterlony plates. The unlabeled well in the center of Fig. 1 is anti-frog lens. The precipitate lines appearing between the antiserum well and antigen well were numbered from the antiserum well outward. The fusion of lines indicates similarity of antigens. The large precipitate line of the anti-frog lens-frog lens system nearest to the well (antigen 1) was similar to antigen 1 of the aqueous humor, to the single antigen of the feeding larva supernate (or the late stage larval tail supernate) and possibly to an antigen in the immature oocytes, but this reaction of identy is uncertain due to the weak reaction of this small oocyte antigen. The fusion of lens line 1 with aqueous humor line 1 is shifted towards the aqueous humor well due to the higher concentration of this antigen in the lens supernate. Lens antigens 1, 2 and 3 usually pass into the antiserum \vell after several days; this necessitates changing the contents of the antiserum ~~11 to remove precipitate. Antigen 4 of the lens was similar to brain antigen 3, n-hile it appeared that lens antigen 6 was similar to brain antigen 1. Antigen 6 of the lens was also identical to antigen 2 of the small oocyte 36 --
60173257
Experimental
Cell
Research
21
544
R. A. Flickinger
and G. Stone
supernate. Lens antigen 2, 3, 7, and 8 were not identical with any other organ antigens. Antigen 5 of the lens appears to be the component which is identical to small oocyte antigen 1, and lens antigen 5 was seen to fuse with the retina antigen, but this latter reaction of identity was not entirely certain, due to the weak reaction of the retinal supernate. Small oocyte antigen 1 sho\ved
a reaction of partial identity with lens antigen 6 in some of the agar-plate experiments and the actual identification of the number of the lens antigen (5 or 6) that is identical with the small oocyte antigen is not definite. The reaction of the brain absorbed anti-frog lens serum with the small oocyte supernate in the precipitin reactions is the best evidence for a common lenssmall oocyte supernate antigen which is not present in the brain supernatc. In order to determine if the small oocyte supernate antigen bearing the lens determinate groups is a major or minor antigen, the antiserum to small oocyte supernate was utilized in agar-diffusion plates in the manner illustrated in Fig. 3. The three lines that appear in the small oocyte supernate-anti frog lens serum system cross over the three lines that appear in the small oocyte supernate-anti small oocyte supernate system and hence they arc not identical. It seems that the components of the small oocyte supernate which react with anti-frog lens serum are not the major antigenic components of the preparation. Anti-frog lens serum was used in combination with anti-testis serum in the Experimental
Cell
Research
21
Lens antigen
localization
manner illustrated in Fig. 4. The antigens in the small oocyte supernatant and the aqueous humor which react with the anti-testis serum are dissimilar to the antigens in these fractions which react with anti-frog lens as shown by the crossing of the antigen-antibody precipitate lines. DISCUSSION
Ten Cate and Van Doorenmaalen [12] \vere able to detect lens antigen serologically in frog and chick embryos at the time the lens placode formed, but not before then. However, there remained a possibility that lens antigen might be present at stages prior to the formation of the lens placode since Flickinger, Levi and Smith [4] found that an anti-cattle lens serum would react with a centrifugal supernate fraction prepared from mature frog oocytes. Another possibility is that lens antigen may be present in other areas than the lens forming regions. Flickinger ef al. [4] found that an anti-cattle lens swum would react with centrifugal supernates of both the anterior and posterior halves of 69 hour chick embryos, and Flickinger [Z] discovered that this antisertim would react nith supernates of the heads and trunks of hatched frog larvae. Van Doorenmaalen [13] utilized a fluorescein-labeled anti-adult lens serum and found uniform staining throughout the chick embryo at 5 days of development and earl&. After 5 days the reaction was specific for the lens. Clayton [2] obtained fluorescein-labeled anti-lens reactions with various parts (optic cup, brain, epidermis) of the early mouse embryo. Langman et ~1. [li] applied anti-lens sera to explants of early chick embryos cultured in vitro and observed not only cytolysis of presumptive lens epidermis up to the 17 somite stage, but also cytolysis of optic cup, brain and all the ectoderm at early stages of development with high concentrations of antiserum . After the 17 somite stage the lens antisera were without effect upon the embryos. This same author [5] localized lens antigens in the pigmented layer of the retina and iris of the hatched chick and postulated that thcsc antigens might be “the biochemical substrate for lens regeneration”. Our results provide evidence in support of the idea that proteins lvith lens-determinate groups are present in immature frog oocytes, head and trunk regions of neurulae, throughout the head region of early tailbuds, larval tails, aqueous humor and the iris-pigmented retina. The fact that brain-absorbed cattlc lens antiserum reacts with these antigen preparations makes it improbable that the reactions are due to combining groups other than those of the Icns. In other wwrds, the reaction is due to lens-like combining groups, not frog-like combining groups. Two major antigens of frog lens Experimental
Cell Research
21
R. A. Flickinger
and G. Stone
are common to frog brain, hut absorption with brain supernate does not remove activity towards the other antigens listed above. In attempting to correlate the presence of lens antigen (or protein with lens-determinate groups) with the determination of the lens and the subsequent differentiation of the lens, it seems that only in the latter case is the answer completely clear. Ten Cate and Van Doorenmaalen [la] first showed an increase in lens antigen at the time the lens placode forms and it is apparent, as might be expected, that actual differentiation entails an increase in lens-antigen. If one considers the stage 19 lens placode as determined, but not differentiated, then determination also is associated with an increase in lens antigen. Our evidence does not support the contention that the amount of lens antigen increases in the earlier stages of lens determination. Our attempt to demonstrate an increase in lens antigen in embryos raised at lO”C, as compared to those raised at 25”C, failed. Also the fact that the optic cup, and overlying ectoderm area, of stage 16 tailhuds, has no greater reaction with anti-frog lens serum than the surrounding areas of the head, argues that during the earlier stages of lens determination, beginning at the neurula stage, there is no increase in lens antigen nor specific lens antigen localization in the lens forming region. The data suggest the speculation that the presence of an antigen with lens specific determinate groups in immature oocytes, neurulae and probably the retina-iris is in some manner a reffection of latent lens forming capacity and that this antigen is not present in tissues which have lost lens competence. SUMMARY
1. By the use of the precipitin method, utilizing brain absorbed anti-frog and anti-cattle lens sera, antigens with lens-determinate groups were detected in immature frog oocytes, neurulae, the tails of late-stage feeding larvae, the pigmented retina-iris and aqueous humor of adult frog eyes. 2. There was no difference noted in the intensity of the precipitin reaction, with brain absorbed anti-frog lens, comparing embryos raised to the neurula stage at 10°C or at 25°C. The heads of both of these temperature groups showed a stronger reaction with the anti-lens serum than did the trunk fractions. At stage 16 the three regions of the head (optic cup, optic vesicle and oral sucker area) all react equally \vell with anti-frog lens serum. 3. Agar diffusion plates indicated the presence of eight frog lens antigens. One of these was similar to an antigen present in early stage feeding larvae, tails of late stage feeding larvae and the aqueous humor. Two other lens Experimenfal
Cell Research
21
Lens antigen
localization
547
antigens showed reactions of identity with two brain antigens and one of these common lens-brain antigens was also found in the immature oocytes. Yet another lens antigen was similar to a second small oocyte supernate antigen. The antigens in the immature oocytes which react with the antifrog lens serum are different from the antigens in this preparation which react with an anti-testis serum and an antiserum to small oocyte supernatc itself. REFERENCES R., Proc. Sac. Exptl. Biol. Xed. 79, 319 (1952). M.. Nature 174. 1059 (1954). R. k., Biol. Bull. 115, ZOl (1658). R. A., LEVI, E. and SMITH, A. E., Physiol. Zoot. 28, 79 (1955). j., Anat: Record 133, 301 (1959). J., SCHALEKAMP, M., KUYKEN, hf. and VEEN, R., Acta Morphol. Neerl. Stand. 1, 142 (1956). LIEDKE, K., J. Ezpft. Zool. 90, 331 (1942). REYER, R. W., Quart. Reu. Biol. 29, 1 (1954). --J. Exptl. Zool. 138, 505 (1958). ~ ibid. 139, 137 (1958). TES CATE, G., The intrinsic development of amphibian embryos. North Holland Pub. Co., Amsterdam, 1953. TEN CATE, G. and VAN DOORENMAALEN, IV. .J., Proc. Koninkl. Nederland. dkad. Wetenschap. 53, 3 (1950). \:AS DOORENMAALEN, W. J., Acta Morphol. Neerl. Stand. 2, 1 (1958).
1. BJ~RKLUND, 2. CLAYTON. 3. FLICKIN~ER, 4. FLICKINGER, 5. LANGMAN, 6. LANGMAN,
7. 8.
9. 10. 11. 12. 13.
R.
Experimental
Cell
Research
21
Cell Research
21, 541-547
LOCALIZATION
541
(1960)
OF LENS
ANTIGENS
FROG
of Zoology,
State
Received
DEVELOPING
EMBRYOS
R. A. FLICKINGER Department
IN
and G. STONE University
December
of Iowa,
Zowa,
U.S.A.
12, 1959
1~ is of some interest to learn if proteins with organ specific combining groups are present in the embryo prior to the differentiation of the organ. The lens is a particularly good organ to use in a serological investigation in relation to embryonic development [la] since it has some degree of organ specificity and there is information from biological experiments concerning the time period when certain tissues can form lenses [‘i, 8, 9, 101.
MATERIALS
AND
METHODS
Antisera were obtained to centrifugal supernates (10,000 xg) of homogenates of frog lenses (Rana pipiens), cattle lenses, immature frog ovaries and frog testes. The homogenates used in preparing injection and test antigens were prepared with an equal volume of tissue and 0.9 per cent NaCl containing merthiolate at a l/10,000 concentration. Protein concentrations of the test antigen preparations were ascertained by nitrogen determinations (Nesslerization) and the use of the factor 6.25. The antisera were produced by injections given every other day to young 6-8 lb. New Zealand rabbits. The injection schedule consisted of intravenous injections of 0.5, 1.0, 2.0, 3.0 and 1.0 ml with a 3.0 ml intraperitoneal injection given with the last intravenous injection. Recall injecstions, consisting of a 1.0 ml intravenous and a 3.0 ml intraperitoneal injection, were given three weeks after the completion of the first injection series. The animals were bled 6 days after the recall injections. Ouchterlony agar plate diffusion experiments were carried out according to the methods of Bjiirklund [l]. RESULTS Precipitin reactions.-The frog lens antiserum was not specific and also reacted against serum and other organs, while the anti-beef lens serum shovved a weak cross reaction with a frog brain supernatc. However, after absorption of one volume of the anti-frog lens serum with 24 volumes of 1 This investigation P’oundation.
was
supported
by
a research
grant
(6139)
from
the
Experimental
National
Science
Cell Research
21
542
R. A. Flickinger
and G. Stone
brain supcrnate in four absorptions, and similar absorption of the anticattle lens serum, these antisera did not react against centrifugal supernates of homogenates prepared from brain, kidney, liver, heart, muscle, stomach, skin and testes; nor did they react with frog serum. The absorbed anti-frog lens serum reacted with a l/SO,000 dilution of the frog lens antigen, which contained 1.25 ,~ugprotein/ml. The unahsorhed anticattle lens serum reacted w?th a 1 /GO,000 dilution of the frog lens antigen, xvhich contained 1.6 ,~ug protein/ml. Roth the anti-frog and anti-cattle lens sera reacted against a l/l0 dilution of frog lens antigen at an antiserum dilution of at least l/10,000. 13oth the frog and the cattle lens antisera, after having been absorbed hvith the brain supernate, reacted positively with the centrifugal supernate of the immature oocytes (small oocyte supernate) at a l/20 dilution. This contained 560 pug protein/ml. Dilution of the two antisera to a 1 jl0 concentration aholished activity. It is known that lens determination occurs hy the neurula stage in Runn esczrlenfa embryos raised at 10-l ‘L”C, hut at 25°C the optic vesicle must play a role in lens determination [l 11. In our experiments two groups of Rnnrr pipiens embryos \\-cre raised to the late neurula stage (Shurn\\-ag stage 15) at two difrerent temperatures (10’ and 25’C). Thcsc embryos \vcre surgically separated into head and trunk regions and centrifugal supernatcs QY’~C’ reacted against the anti-lens sera in precipitin reactions. The head and trunk fractions of the embryos raised at 10’ and ‘25°C reacted equally xvell with absorbed anti-frog lens serum, but did not react with anti-cattle lens strum. Positive reactions were obtained with l/l0 (hut not l/20) antigen dilutions of the 10” and 25°C head fractions which contained approximately 2,000 ,LL~ protein/ml. The trunk antigens (10’ and 25°C) did not react at a l/l0 antigen dilution; ho\vcvcr, their protein concentration was about half that of the head fractions. The prospective otic capsule, optic vesicle and oral sucker areas were’ one hundred early tailbud Rnnrt pipiens extirpated from approximately embryos at Shumway stage 16, prior to any morphological specialization of these structures. The centrifugal supcrnatcs from these areas were utilized as test antigen preparations and each reacted within t\\-enty minutes in precipitin reactions with anti-frog lens serum that had been partially absorbed three times with brain supernate. They were inactive against the anti-frog serum that had been completely absorbed by four brain supernate absorptions. This inactivity is ascribed to further dilution of the antiserum. The tails of late stage feeding larvae of Rnnrr pipiens (A-80 mm) were Experimental
Cell Research
21
Lens antigen
localization
prepared anti-frog
as centrifugal supernates and found to react with the brain absorbed antigen dilution of Rana and anti-cattle lens sera. The limiting pipiens larval tail fraction which reacted with the brain absorbed anti-frog lens serum was l/10. This contained 212 pg protein/cc. The brain absorbed anti-frog lens serum gave a positive reaction at a l/l0 dilution of antiserum. A ten fold dilution of anti-cattle lens serum was inactive. The aqueous humor and the iris-pigmented retina were removed from the eyes of adult frogs (Rana pipiens) and from two species of salamanders (Triturus (Taricha) torosus and T. rivularis). The anti-frog lens serum reacted with a l/25 dilution of frog aqueous humor (51 pg protein/ml) and a l/l0 dilution of salamander aqueous humor. The frog lens antiserum was still active against undiluted frog aqueous humor (275 ,ug protein/ml) at a l/l0 antiserum dilution. Only the undiluted anti-cattle lens serum reacted with frog aqueous humor. The undiluted iris-pigmented retina centrifugal supernate (1,250 lug protein/ml) reacted with the anti-frog lens and anti-cattle lens sera. A l/l0 dilution of frog lens antiserum could still react with the irisretina test antigen. Agar plate diffusion reactions.-In order to determine if the antigens in the immature oocytes, brain, serum, whole feeding larvae and the larval tail were similar or dissimilar to those in the aqueous humor, retina and the lens itself, unabsorbed anti-frog lens serum was utilized in a number of agar plate diffusion tests. The results are shown in Figs. 1 and 2. These drawings represent a combination of the results of a large series of Ouchterlony plates. The unlabeled well in the center of Fig. 1 is anti-frog lens. The precipitate lines appearing between the antiserum well and antigen well were numbered from the antiserum well outward. The fusion of lines indicates similarity of antigens. The large precipitate line of the anti-frog lens-frog lens system nearest to the well (antigen 1) was similar to antigen 1 of the aqueous humor, to the single antigen of the feeding larva supernate (or the late stage larval tail supernate) and possibly to an antigen in the immature oocytes, but this reaction of identy is uncertain due to the weak reaction of this small oocyte antigen. The fusion of lens line 1 with aqueous humor line 1 is shifted towards the aqueous humor well due to the higher concentration of this antigen in the lens supernate. Lens antigens 1, 2 and 3 usually pass into the antiserum \vell after several days; this necessitates changing the contents of the antiserum ~~11 to remove precipitate. Antigen 4 of the lens was similar to brain antigen 3, n-hile it appeared that lens antigen 6 was similar to brain antigen 1. Antigen 6 of the lens was also identical to antigen 2 of the small oocyte 36 --
60173257
Experimental
Cell
Research
21
544
R. A. Flickinger
and G. Stone
supernate. Lens antigen 2, 3, 7, and 8 were not identical with any other organ antigens. Antigen 5 of the lens appears to be the component which is identical to small oocyte antigen 1, and lens antigen 5 was seen to fuse with the retina antigen, but this latter reaction of identity was not entirely certain, due to the weak reaction of the retinal supernate. Small oocyte antigen 1 sho\ved
a reaction of partial identity with lens antigen 6 in some of the agar-plate experiments and the actual identification of the number of the lens antigen (5 or 6) that is identical with the small oocyte antigen is not definite. The reaction of the brain absorbed anti-frog lens serum with the small oocyte supernate in the precipitin reactions is the best evidence for a common lenssmall oocyte supernate antigen which is not present in the brain supernatc. In order to determine if the small oocyte supernate antigen bearing the lens determinate groups is a major or minor antigen, the antiserum to small oocyte supernate was utilized in agar-diffusion plates in the manner illustrated in Fig. 3. The three lines that appear in the small oocyte supernate-anti frog lens serum system cross over the three lines that appear in the small oocyte supernate-anti small oocyte supernate system and hence they arc not identical. It seems that the components of the small oocyte supernate which react with anti-frog lens serum are not the major antigenic components of the preparation. Anti-frog lens serum was used in combination with anti-testis serum in the Experimental
Cell
Research
21
Lens antigen
localization
manner illustrated in Fig. 4. The antigens in the small oocyte supernatant and the aqueous humor which react with the anti-testis serum are dissimilar to the antigens in these fractions which react with anti-frog lens as shown by the crossing of the antigen-antibody precipitate lines. DISCUSSION
Ten Cate and Van Doorenmaalen [12] \vere able to detect lens antigen serologically in frog and chick embryos at the time the lens placode formed, but not before then. However, there remained a possibility that lens antigen might be present at stages prior to the formation of the lens placode since Flickinger, Levi and Smith [4] found that an anti-cattle lens serum would react with a centrifugal supernate fraction prepared from mature frog oocytes. Another possibility is that lens antigen may be present in other areas than the lens forming regions. Flickinger ef al. [4] found that an anti-cattle lens swum would react with centrifugal supernates of both the anterior and posterior halves of 69 hour chick embryos, and Flickinger [Z] discovered that this antisertim would react nith supernates of the heads and trunks of hatched frog larvae. Van Doorenmaalen [13] utilized a fluorescein-labeled anti-adult lens serum and found uniform staining throughout the chick embryo at 5 days of development and earl&. After 5 days the reaction was specific for the lens. Clayton [2] obtained fluorescein-labeled anti-lens reactions with various parts (optic cup, brain, epidermis) of the early mouse embryo. Langman et ~1. [li] applied anti-lens sera to explants of early chick embryos cultured in vitro and observed not only cytolysis of presumptive lens epidermis up to the 17 somite stage, but also cytolysis of optic cup, brain and all the ectoderm at early stages of development with high concentrations of antiserum . After the 17 somite stage the lens antisera were without effect upon the embryos. This same author [5] localized lens antigens in the pigmented layer of the retina and iris of the hatched chick and postulated that thcsc antigens might be “the biochemical substrate for lens regeneration”. Our results provide evidence in support of the idea that proteins lvith lens-determinate groups are present in immature frog oocytes, head and trunk regions of neurulae, throughout the head region of early tailbuds, larval tails, aqueous humor and the iris-pigmented retina. The fact that brain-absorbed cattlc lens antiserum reacts with these antigen preparations makes it improbable that the reactions are due to combining groups other than those of the Icns. In other wwrds, the reaction is due to lens-like combining groups, not frog-like combining groups. Two major antigens of frog lens Experimental
Cell Research
21
R. A. Flickinger
and G. Stone
are common to frog brain, hut absorption with brain supernate does not remove activity towards the other antigens listed above. In attempting to correlate the presence of lens antigen (or protein with lens-determinate groups) with the determination of the lens and the subsequent differentiation of the lens, it seems that only in the latter case is the answer completely clear. Ten Cate and Van Doorenmaalen [la] first showed an increase in lens antigen at the time the lens placode forms and it is apparent, as might be expected, that actual differentiation entails an increase in lens-antigen. If one considers the stage 19 lens placode as determined, but not differentiated, then determination also is associated with an increase in lens antigen. Our evidence does not support the contention that the amount of lens antigen increases in the earlier stages of lens determination. Our attempt to demonstrate an increase in lens antigen in embryos raised at lO”C, as compared to those raised at 25”C, failed. Also the fact that the optic cup, and overlying ectoderm area, of stage 16 tailhuds, has no greater reaction with anti-frog lens serum than the surrounding areas of the head, argues that during the earlier stages of lens determination, beginning at the neurula stage, there is no increase in lens antigen nor specific lens antigen localization in the lens forming region. The data suggest the speculation that the presence of an antigen with lens specific determinate groups in immature oocytes, neurulae and probably the retina-iris is in some manner a reffection of latent lens forming capacity and that this antigen is not present in tissues which have lost lens competence. SUMMARY
1. By the use of the precipitin method, utilizing brain absorbed anti-frog and anti-cattle lens sera, antigens with lens-determinate groups were detected in immature frog oocytes, neurulae, the tails of late-stage feeding larvae, the pigmented retina-iris and aqueous humor of adult frog eyes. 2. There was no difference noted in the intensity of the precipitin reaction, with brain absorbed anti-frog lens, comparing embryos raised to the neurula stage at 10°C or at 25°C. The heads of both of these temperature groups showed a stronger reaction with the anti-lens serum than did the trunk fractions. At stage 16 the three regions of the head (optic cup, optic vesicle and oral sucker area) all react equally \vell with anti-frog lens serum. 3. Agar diffusion plates indicated the presence of eight frog lens antigens. One of these was similar to an antigen present in early stage feeding larvae, tails of late stage feeding larvae and the aqueous humor. Two other lens Experimenfal
Cell Research
21
Lens antigen
localization
547
antigens showed reactions of identity with two brain antigens and one of these common lens-brain antigens was also found in the immature oocytes. Yet another lens antigen was similar to a second small oocyte supernate antigen. The antigens in the immature oocytes which react with the antifrog lens serum are different from the antigens in this preparation which react with an anti-testis serum and an antiserum to small oocyte supernatc itself. REFERENCES R., Proc. Sac. Exptl. Biol. Xed. 79, 319 (1952). M.. Nature 174. 1059 (1954). R. k., Biol. Bull. 115, ZOl (1658). R. A., LEVI, E. and SMITH, A. E., Physiol. Zoot. 28, 79 (1955). j., Anat: Record 133, 301 (1959). J., SCHALEKAMP, M., KUYKEN, hf. and VEEN, R., Acta Morphol. Neerl. Stand. 1, 142 (1956). LIEDKE, K., J. Ezpft. Zool. 90, 331 (1942). REYER, R. W., Quart. Reu. Biol. 29, 1 (1954). --J. Exptl. Zool. 138, 505 (1958). ~ ibid. 139, 137 (1958). TES CATE, G., The intrinsic development of amphibian embryos. North Holland Pub. Co., Amsterdam, 1953. TEN CATE, G. and VAN DOORENMAALEN, IV. .J., Proc. Koninkl. Nederland. dkad. Wetenschap. 53, 3 (1950). \:AS DOORENMAALEN, W. J., Acta Morphol. Neerl. Stand. 2, 1 (1958).
1. BJ~RKLUND, 2. CLAYTON. 3. FLICKIN~ER, 4. FLICKINGER, 5. LANGMAN, 6. LANGMAN,
7. 8.
9. 10. 11. 12. 13.
R.
Experimental
Cell
Research
21