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The inhibition of lens-inducing capacity of the optic vesicle with adult lens antisera
DEVELOPMENTAL
The
BIOLOGY,
2, 155-172
(
1960)
Inhibition of Lens-inducing Optic Vesicle with Adult M.
WILLIAM Department
of
Anutomy,
CLARKE’
Capacity of the Lens Antisera
AND
IRA
University of North North Carolina
Accepted
Janzrmy
FOWLER” Curolina,
Chapel
tlill,
26, 1960
INTRODUCTION
Several investigators (Alexander, 1937; van Deth, 1940; McKeehan, 1951; Langman, 1956) have pointed out that in chick embryos the lens originates from head ectoderm in contact with, and under the inductive influence of, the optic vesicle. Induction occurs during the period (9-21 somites) the optic vesicle adheres closely to the presumptive lens ectoderm ( McKeehan, 1951). Prior to the 9-somite stage, the presumptive lens ectoderm can be readily separated from the optic vesicle. Separation of the two tissues by mechanical means is extremely difficult at lo-21 somites of age. After 21 somites of age, the lens ectoderm attains capacity of self-differentiation. The presence of lens antigens in the embryonic chick lens has been demonstrated by serological methods (see Woerdeman, 1955; van Doorenmaalen, 1958, for review of the literature). Using fluoresceinconjugated antilens sera, van Doorenmaalen (1958) was unable to demonstrate a clear-cut localization of lens antigens prior to 5 days of incubation. With the precipitin method, ten Cate and van Doorenmaalen (1950) f ound adult lens antigens in the extract of lens vesicles of chick embryos as early as 58 hours’ incubation. Langman et al. (1957) reported evidence of the presence of precursors of lens proteins in 5-17 somite stages. This observation was based upon the cytolysis of presumptive lens ectoderm exposed, in vitro, to antiadult lens sera. 1 Aided by a Medical Student Fellowship from the National Foundation. 2 This investigation was supported by a senior Research Fellowship (SF-93-C from the Public Health Service. L55
)
156
WILLIAM
M.
CLARKE
AND
IRA
FOWLER
On the basis of an experiment (described more fully in the discussion of this paper) Woerdeman (1955) suggests that precursors in the brain and optic vesicles of early embryos may be involved in lens induction by reacting with precursors in the presumptive lens ectoderm. In such a system, precursors from both sources would be essential to the inductive process. If the precursors in the optic vesicle are similar to adult lens antigens, it might be possible to bind these precursors with adult lens antisera and thus prevent their activity in the induction process. The purpose of this investigation was to study the effect of adult lens antisera upon the inducing capacity of the optic vesicle by exposing the isolated optic vesicle to the antisera and subsequently testing its capacity to induce a lens in competent ectoderm. METHODS
1. Preparation
of Antisera
Adult chicken lenses obtained from freshly killed chickens were dissected free of the lens capsule and homogenized with an equal volume of physiological saline in. an all-glass (ten Broeck) homogenizer at 0-4O C. The homogenate was centrifuged at 14,000 x gravity at O-2” C for 15 minutes in a Servall centrifuge. Total nitrogen determinations (method of Lanni et aZ., 1950) showed that the supernatant contained approximately 100 mg of protein per milliliter. Two New Zealand white rabbits were immunized with this supernatant, each receiving five injections of the antigen; the injections were given on alternate days over a lo-day period. Each rabbit received a total of 288 mg of protein. Fourteen days after the initial injection of the antigen, the sera were collected and pooled. The sera gave a precipitate with all dilutions of the antigens up to and including one containing 0.001 mg of protein per milliliter. The crude sera were fractionated by the ammonium sulfate technique; the precipitated globulin was resuspended in one-fourth of the original volume in physiological saline buffered at pH 7.0 and was then stored at O-40 C until used. The interface precipitin test was the same as indicated with original crude antisera. Normal rabbit sera were fractionated similarly for use in the controls. 2. Fluorescent Normal
rabbit
Procedures globulin
and antilens globulin
were
conjugated
with
ANTISERA
ACTION
IN
LENS
157
INDUCTION
fluorescein isocyanate (Sylvana Chemical Company) according to the technique of Coons and Kaplan (1950). The fluorescent conjugate was employed as a histochemical stain for lens antigens in sections of embryos of 7 somites to 5 days of age. The yellow-green fluorescence on a black background marked the site of antigen localization in the section. Embryos were prepared according to the technique of Davidson ( 1945). Following rehydration one drop of the fluorescent globulin was placed on the section; after this it was incubated in a humidified chamber at 37O C for 30 minutes. Excess globulin was removed by washing in diethyl barbiturate buffer at pH 8.1 for 20 minutes. Sections were mounted in a mixture of buffered saline and glycerol and examined under the fluorescent microscope (Leitz ortholux microscope with ultraviolet illuminator). Photomicrographs were prepared using plus X film. Sections adjacent to the one stained with fluorescent antilens sera were employed as controls. These included: (1) a section stained with fluorescent normal rabbit globulin; (2) a section treated with unconjugated antilens globulin followed by the fluorescent antilens globulin; and (3) one unstained section.
#199+ 10% horse sera E= ‘120 anti-adult lens 6ero C= kzo normal rabbit ?,er(l I8 hours
%I99
t 10% horse
sere
FIG.
Procedure
1.
3. Effect of Antilens
Earle’s solutmn 30 minutes
7-somite
used
in
in vitro
studies
(see
text
for
ectoderm
details).
Sera in Vitro
The operative procedure is outlined in Fig. 1. Optic vesicles of embryos of P7 somites (Hamburger and Hamilton, 1951, stages 8 and 9) were removed, free of ectoderm, with tungsten needles prepared according to Dossel (1958). Care was taken to include a minimum of brain with the optic vesicle, although mesenchyme was not en-
158
WILLIAM
M.
CLARKE
AND
IRA
FOWLER
tirely removed. Each optic vesicle was transferred to a glass microculture slide containing 19 parts artificial medium 199 (Microbiological Associates) with 10% horse serum added and 1 part of adult-lens antisera (globulin fraction containing 0.001 mg of protein per milliliter). The vesicle was allowed to settle to the bottom of the concavity. TWO microculture slides were placed in a petri dish on moistened filter paper (a modification of the culture technique of Fell and Robison, 1929) and -incubated for a period of 18 hours at 38’ C. After 18 hours of culture, the optic vesicles were removed from the culture medium and washed for 30 minutes in at least three changes of Earle’s solution to remove as much of the lens antisera as possible. The vesicles were then returned to individual culture slides and incubated until strips of ectoderm could be prepared for the next phase of the experiment. Relatively large strips of ectoderm were removed from embryos of 4-7 somites that had been stained lightly with Nile blue sulfate. The ectoderm removed included the presumptive lens ectoderm anteriorly, and extended posteriorly to approximately the future level of the otocyst. Laterally, it included all the embryonic area. The medial incision was immediately lateral to the brain, The piece of ectoderm was washed in Earle’s solution and transferred immediately to a culture dish containing an optic vesicle. The optic vesicle, during the B-hour culture period, typically rounded up into a ball, which facilitated its enclosure in the ectoderm. The ectoderm strip was spread out on the bottom of the glass dish with surface side down, and the round optic vesicle was moved on top of the ectoderm. The ectoderm and vesicle were rolled in such a way as to wrap the vesicle completely within the ectoderm. The ectoderm frequently was overlapped. A glass needle was run through the combined tissues to attempt to ensure direct contact between the two layers (see Fig. 1). The needle was removed the following day (approximately 12 hours later ) . The combined optic vesicle-ectoderm cultures were incubated for a period of 3-4 days. The cultures were then fixed in Rouin’s solution, sectioned at 7 p., and stained with hematoxylin. The same experimental procedure was followed in the controls, using the globulin fraction of normal rabbit sera instead of adult-lens antisera during the 18-hour culture period prior to enclosing the optic vesicle in ectoderm. Optic vesicles with the overlying ectoderm intact were cultured in
ANTISERA
ACTION
IN
LENS
INDUCTION
159
19 parts medium 199 + 10% horse serum and 1 part adult lens antisera in procedure similar to that used by Langman et al. (1957) to determine if the antisera would prevent the formation of a lens at the concentration used in these experiments. No lenses formed in the presence of the antisera. As an additional control, sections of the hindbrain and overlying ectoderm, which included the presumptive otocyst, were grown in the above medium containing a 120 dilution of the antisera to determine whether or not the antisera used were exerting a specific effect upon differentiation of the lens or a generalized effect that would prevent differentiation of the otic vesicle. 4. In Vivo Studies
Optic vesicles were removed free of ectoderm from 4-7 somite embryos and cultured for 18 hours as in the in vitro studies previously described. In the experimental series the liquid medium contained a 1:20 dilution of anti-adult lens globulin; in the control series a similar dilution of normal rabbit globulin was used. At the end of the 1% hour culture period the vesicles were washed in Earle’s solution and implanted in host embryos P7 somites of age. The implant site was immediately posterior to the host optic vesicle. Care was taken to locate the vesicle between the ectoderm and underlying mesoderm so that there would be little or no mesoderm between the vesicle and ectoderm. After the implant was in place, the eggs were sealed and incubated for an additional period of 3-4 days. The embryos were fixed in Bouin’s solution, and the region of the graft was sectioned serially and stained in hematoxylin. RESULTS
Optic vesicles extirpated, free of ectoderm, from embryos of 4-7 somites and cultured on glass in liquid medium evidenced little growth during the initia1 H-hour culture period. The cut edges of the vesicle tended to heal together, so that after 18 hours, the vesicle was round. Thus, when the vesicle was wrapped in ectoderm, the sheet of ectoderm was in direct contact with the presumptive retina (the part normally in contact with presumptive lens ectoderm). Len9
lntl~rc+on
in Vitro
Treatment of the optic vesicles for a period of 18 hours with adult
160
WILLIAM
M.
CLARKE
AND
IRA
FOWLER
lens antisera had no detectable morphological effect in comparison with vesicles similarly cultured in normal rabbit sera. Some cytolysis occurred in both the experimental series in which the optic vesicles were exposed to the adult lens antisera (Figs. 2-4, Plate I) and in the
PLATE I C&l. The optic vesicles were treated with adult-lens antisera for 18 prior to enclosure in ectoderm. FIG. 5. The optic vesicle was treated with rabbit sera for 18 hours prior to enclosure in ectoderm. FIGS. 2-5. Exwere cultured 4 days after enclosure in ectoderm. Magnification: X 100. 2. The ectoderm was lost during the culture period. The optic vesicle increased in size, but there was no evidence of “cup” formation. 3. The optic vesicle (0.0.) maintained close contact with the ectoderm but no lens formed. A lens formed in only 2 of 34 similar explants. 4. The optic vesicle ( o.u.) is widely separated from the ectoderm No lens formed in this type of explant. 5. The lens is well developed, although there is considerable necrosis the optic vesicle. A lens differentiated in 35 of 43 explants of this type.
FIGS.
hours normal plants FIG. (0.0.) FIG.
(ect.), FIG.
(ect.). FIG.
within
ANTISERA
ACTION
IN
LENS
161
INDUCTION
control series, in which the optic vesicles were exposed to normal rabbit sera (Fig. 5). Cytolysis within the optic vesicle did not appear to affect directly its capacity to induce a lens. Cytolysis within the optic vesicle was, perhaps, a factor in preventing maintenance of contact between the ectoderm and the optic vesicle. The most striking difference between the two series was in the differentiation of the ectoderm in response to contact with the optic vesicles. Results of both the control series and the experimental series are summarized in Table 1. In 14 of the controls and 22 experimentals, the ectoderm
LENS
Denuded Ectoderm Ectoderm Total
DEVELOPMENT
TABLE 1 IN HEAI) ECTODERM WITH OPTIC VESICLES
vesicle not fused with O.V. fused with O.V.
14 9 43 66
0 0 35 35
IN RESPOXSE in Vitro
0 0 81 53
22 5 34 61
TO CONTACT
0 0 2 2
0 0 6 3
a Prior to enclosure in head ectoderm the optic vesicles (o.v.) were cultured 18 hours in a 1: 20 dilution of normal rabhit serum in Medium 199 plus 10% horse sera. b Prior to enclosure in head ectoderm, the optic vesicles were cultured 18 hours in a 1:20 dilution of the anti-adult lens sera in Medium 199 plus 10% horse sera.
was lost in the process of handling the vesicles. These cultures (see Fig. 2) were the result of experimental error. Since there was no opportunity for lens induction to occur in these cases, they furnish no information as to the lens-inducing capacity of these vesicles. In 9 of the controls and 5 of the experimental cultures, the ectoderm enclosed, but did not maintain direct contact with, the optic vesicles. An example of this type of culture is shown in Fig. 4. The ectoderm is separated from the optic vesicle by considerable space, which was presumably filled with culture medium. Induction of a lens does not occur under these conditions ( McKeehan, 1951; Langman, 1956), although McKeehan (1958) has been able to obtain lenses in viva without direct contact between optic vesicle and presumptive lens ectoderm. Since the above cases (denuded vesicles and cultures in
162
WILLIAM
M.
CLARKE
AND
IRA
FOWLER
ANTISERA
ACTION
IN
LENS
INDUCTION
163
which ectoderm did not fuse with the optic vesicle) failed to provide opportunity for lens induction to occur, they are eliminated from consideration in the evaluation of the data regarding the effect of the anti-adult lens sera upon the lens-inducing capacity of the optic vesicle. The remaining cultures (43 of the control series and 34 of the experimental series) are useful in assessing the lens-inducing capacity, since in these cases the ectoderm established and maintained contact with the optic vesicle (see Table 1). In 35 of 43 cultures in which the optic vesicles were exposed 18 hours to a 1:20 dilution of normal rabbit sera prior to enclosure in competent ectoderm, a lens was induced in ectoderm (Fig. 5). This is contrasted with the cultures in which the optic vesicles were treated with a 1:20 dilution of antiadult lens sera prior to enclosure in competent head ectoderm. In these cases, illustrated in Fig. 3, a lens developed in only 2 of 34 (6%) cultures, even though extensive area of contact between ectoderm and vesicle was demonstrated histologically. The lenses produced were in the form of placodes similar to that shown in Fig. 8 ( Plate II ) . In cultures in which the ectoderm was overlapped extensively, as is shown in Fiq. 6. the optic vesicles failed to grow and excessive cytolysis occurred in both the control and experimental series. Apparently, exchange between the cells of the optic vesicle and surrounding
FIGS.
FIG. dcrm necrotic, cultured FIG.
(h.h.)
PLATE II 6-9. Magnification: x 100. 6. The optic vesicle is completely enclosed in a double layer of ectoas a result of extensive overlan of the ectoderm. The optic vesicle is although it is a control vesicle treated with normal rabbit sera. Explant 3 days after the optic vesicle was enclosed in ectoderm. 7. Explant included a lateral half of the primordium of the hindbrain and the adjacent ectoderm. An otic vesicle (ot.u.) developed in the
ectodcrm. FIG. 8. The lens is in the form of a placode and is separated from the optic vesicle (0.0.). Presumably, the lens ectoderm was in contact with the optic vesicle during induction. The optic vesicle was a control vesicle exposed to normal rabbit sera and cultured 4 days after enclosure in ectoderm. FIG. 9. Exulant included the entire primordium of the hindbrain and the adjacent ectoderm. An otic vesiclr (ot.0.) developed in the ectoderm. The nervous tissue is completely enclosed in ectoderm and contains more necrotic cells than does the nervous tissue (lateral half of hindbrain) shown in Fig. 7.
164
WILLIAM
M.
CLARKE
AND
IRA
FOWLER
PLATE III FIGS. 10-12. Magnification: x 50. FIG. 10. The donor optic vesicle (d.o.o.) was exposed to normal rabbit sera for 18 hours prior to implantation in the host embryo. A lens (d.2.) was induced in the host ectoderm. This lens is smaller than the lens (h.Z.) of the host embryo. FIG. 11. Procedure was similar to that followed in specimen shown in Fig. 10. The lens (d.l.) induced in the host embryo by the donor optic vesicle (d.o.u.) is comparable in size and differentiation to that of the host lens (h.l.).
ANTISERA
medium was insufficient of ectoderm. Lens Induction
ACTION
IN
to maintain
LENS
165
INDUCTION
cells enclosed in a double
layer
in Vivo
By implanting optic vesicles in host embryos, an attempt was made to determine whether the failure of the optic vesicles exposed to antiadult lens sera in vitro was, perhaps, due to relatively poor conditions for lens differentiation. Mortality of the host embryos was high, and the optic vesicle slipped out of the implantation site in a number of specimens. In 3 of 5 specimens in which control vesicles were implanted, contact between donor optic vesicle and host ectoderm was demonstrated histologically. In each of these 3 cases a lens developed from the host ectoderm (see Figs. 10 and 11, Plate III). In 5 of 7 specimens in which experimental vesicles (previously exposed to anti-adult lens sera) were implanted, contact between donor optic vesicle and host ectoderm was observed as illustrated in Fig. 12 (Plate III). No lens differentiated in the host ectoderm in response to the influence of these optic vesicles; these results conform to the findings in the in vitro studies. Effect of Anti-adult
Lens Sera upon Formation of Otic Vesicle
To test whether the antilens sera would have a generalized effect upon induction of other head structures, its capacity to block the formation of the otic vesicle was studied. The otic vesicle develops from head ectoderm under the inductive influence of the hindbrain (Waddington, 1937) and adjacent neural crest (Szepsenwol, 1933). Induction is probably complete by about 4 somites of age. Primordia of hindbrain (either whole or a lateral half) together with the overlying ectoderm from embryos younger than 4 somites were cultured in a medium containing a 1:20 dilution of the anti-adult lens sera. In each of 16 cases (see Figs. 7 and 9) the otic vesicle was formed even in cultures in which the hindbrain tended to cytolyze. Thus it is evident that the antisera did not prevent induction of the otic FIG. 12. The sera for 18 hours the host ectoderm host ectoderm.
donor optic vesicle (d.o.u.) was exposed prior to implantation in the host embryo. even though the optic vesicle established
to anti-adult lens No lens formed in contact with the
ANTISERA
ACTIOK
IN
LENS
INDUCTION
vesicle at the concentration used in this study. The action appears be specific in its effect upon eye structures. Normal
Embryos
Stained
with
Fluorescent
167 to
Antisera
In an attempt to determine why there was apparently a specific effect of the antilens sera upon the lens-inducing capacity of the optic vesicles, a series of normal embryos from 7 somites to 5 days of age were stained with anti-adult lens sera conjugated with fluorescein isocyanate. Sections of selected stages stained with the fluorescent antisera are illustrated in Figs. 13-16 (Plate IV). These sections were stained with hemotoxylin following the fluorescent study and are shown in Figs. 17-20 (Plate IV). In normal S-day embryos, the lens shows an intense fluorescence following treatment with the fluorescent antisera. The results at this age appear to be similar to those of van Doorenmaalen ( 1958). In younger embryos, van Doorenmaalen found only a generalized nonspecific type staining. Owing, perhaps, to the difference in the fixative used (van Doorenmaalen used acetone; we used Smith’s Fluid), the younger embryos appeared to stain more intensely than was the case in van Doorenmaalen’s careful study. As may be seen in comparing Figs. 13, 14, 15, and 16 the intensity of staining is progressively less in the younger embryos. In embryos of I9 somites (Fig. 14)) at which induction is almost complete, the optic vesicle and presumptive lens ectoderm fluoresce with approximately c~rual intensity. In embryos of 12 somites (Fig. 13), prior to lens PLATE IV FIGS. 13-16. Normal embryos stained with anti-adult lens sera conjugated with fluorescein isocyanate. Microphotographs were made with uniform conditions of lighting, exposure time, and magnification. The degree of lightness in the print reflects the intensity of fluorescence of the specimen. Magnification: x 100. FIG. 13. Embryo of 12 somites. The optic vesicle (0.0.) fluoresces with greater intensity than does the adjacent ectoderm (ect. ) . Embryo of 19 somites. The optic vesicle and adjacent lens ectoderm FIG. 14. fluoresce with approximately equal intensity. The lens of a 4-day embryo. The intensity of fluorescence is greater FIG. 15. than in Figs. 13 and 14, but less than is seen in Fig. 16. FIG. 16. The lens of a 5-day embryo. Fluorescence is localized in the lens and is of greater intensity than in younger stages. FIGS. 17-20. Same as Figs. 13-16; stained with hematoxylin. Magnification: x 100.
168
WILLIAM
M.
CLARKE
AND
IRA
FOWLER
induction, the optic vesicle fluoresces with greater intensity than does the presumptive lens ectoderm. This indicates that there are substances in the optic vesicle prior to lens induction that react with fluorescein conjugated adult lens antisera. The reaction of substances within the presumptive lens ectoderm with the fluorescent lens antisera increases as lens induction proceeds; simultaneously, the reaction of substances within the optic cup with fluorescent lens antisera decreases so that by 5 days this reaction is, as van Doorenmaalen has shown, localized within the lens. DISCUSSION
It is well known that inducing and induced tissues have an intimate association with one another. This is especially well demonstrated in the development of the lens, where certain cytological changes in the presumptive lens ectoderm are shown to occur in response to contact with the optic vesicle. McKeehan (1951) has described in detail the cytological changes in the presumptive lens epithelium, consisting of loss of vacuolization, nuclear orientation, and palisade formation. Weiss (1956) suggests the nuclear orientation may indicate simultaneous molecular orientation at the cell surface. McKeehan (1958) has observed a few instances of lenses induced in ectoderm separated from the optic vesicle by a thin agar membrane. This suggests substances of a diffusible nature may be involved in the lens induction process. Evidence is accumulating that inductors in general are diffusible. The experiments of Lash et al. (1957) demonstrate that neural tube substances can penetrate a porous membrane and cause differentiation of cartilage, although this process may not be induction in the classic sense. The studies of Niu (1956) indicate clearly that the neural inductor with which he is working is diffusible. The appearance of specific antigenic substances has been shown in many instances, to be correlated with morphological events. However, there is little evidence that specific antigens play a causal role in differentiation. Woerdeman (1955) suggests such a role in lens development. Saline extracts of head ectoderm of young axolotl neurulae before the appearance of a lens placode failed to react with antisera to adult lens. The same was found to be true with extracts of young, isolated eye vesicles. The extracts of eve vesicle and presumptive lens ectoderm were mixed and incubated at 370 C
ANTISERA
ACTION
IN
LENS
INDUCTION
169
for 24 hours. This mixture then showed a precipitin reaction with lens antisera. Presumably, new compounds were formed in vitro that reacted with lens antisera. From this it might be inferred that the production of lens antigens results from the interaction between head ectoderm and eye vesicle. Such a system would explain the results of our experiments. If the eye vesicle substances involved in the above process contained groups that react with adult lens antisera, then these substances would be inactivated by reaction with antibodies to lens antigens and induction would fail. In our investigation, the staining of normal embryos with fluorescein-conjugated antisera indicates that there are indeed substances present in the optic vesicle prior to and during the induction process which can react with adult lens antisera. The inhibition of the lens-inducing capacity of the eye vesicle by adult-lens antisera suggests that these substances which react with the antisera are essential to the induction process. The nature of the substances is unknown. Sirlin and Brahma, 1959, have presented evidence of transfer of both small molecules and complex, more specific, molecules or particles from the optic vesicle to the lens during lens induction in Xenopus. Eyecups incubated with C4labeled phenylalanine and subsequently enclosed in ectoderm, in a procedure similar to that used in this investigation, induced lenses in the ectoderm. The tracer was clearly detectable in the developing lens. Eyecup cytoplasm near the lens was depleted of the labeled proteins, whereas labeling of the lens cytoplasm increased. Although this study does not show directly that the labeled proteins are involved in the induction of the lens, the shift of tracer into the lens during the period of induction is certainly suggestive. In our study, the molecules involved are probably not identical with lens proteins. They may merely happen to contain determinant groups capable of reacting with some part of one or more of the antibody molecules. On the other hand, the reaction in the optic vesicle that is evident from the staining of normal embryos with the fluorescein conjugated antibody may be a reaction between the antibody and compounds not involved in lens induction. This appears to be unlikely in view of the fact that treating the optic vesicles with unconiugated antibody virtually eliminated its capacity to induce a lens. The possibility that the action of the antilens sera is a generalized toxic effect rather than a specific reaction with certain compounds within the optic vesicle has not been eliminated conclusively in
170
WILLIAM
M.
CLARKE
AND
IRA
FOWLER
these experiments. However, the antisera did not prevent induction of the otocyst at the same concentration used in treating the optic vesicles. This, together with the fact that there was no detectable morphological effect of the antisera upon the optic vesicles in comparison with the controls, suggests a high degree of specificity in the action of the antisera. Langman et al. (1957) observed unmistakable evidence of an effect of their antilens sera upon the optic vesicle. At concentrations of antilens sera which were considerably higher than that used in this study, several of their preparations showed slight degeneration of the optic cup. The concentration of antisera used in our experiments produced no morphological changes that could be ascribed to the antisera. This was perhaps due to the slow growth medium used (artificial medium 199 with 10% horse sera added). Langman used embryonic extract. In the course of our investigation, cultures of optic vesicles with the overlying ectoderm left in place were exposed in artificial medium 199 to a 1:20 dilution of antilens sera. Typical lenses failed to develop in 100% of the cases. In similar cultures with normal rabbit sera substituted for the antilens sera, lenses developed in 75% of the cultures. The inhibitory action of antilens sera appears to be more effective when used in artificial medium than when added to embryonic extract. SUMMARY
1. Optic vesicles removed, free of ectoderm, from chick embryos of 4-7 somites were explanted for 18 hours in a medium containing a 1:20 dilution of the globulin fraction of anti-adult lens sera. The vesicles were then enclosed in competent ectoderm and cultured 3-4 days in a medium free of the antisera. Induction of a lens occurred in 2 of 61 cases (3%). 2. Induction of a lens resulted in 35 of 66 cases (53%) in the control vesicles cultured in a 1:20 dilution of normal rabbit sera (globulin fraction) and subsequently enclosed in ectoderm. 3. The results of experiments in which optic vesicles were cultured in media containing either antilens sera or normal rabbit sera and subsequently implanted beneath the ectoderm of 4-7 somite embryos conform to the results of the in vitro experiments. 4. A 1:20 dilution of the anti-adult lens sera failed to inhibit development of the otic vesicle. 5. Sections of selected stages of normal embryos were stained
ANTISERA
ACTION
IN
LENS
171
INDUCTION
with antilens sera conjugated with fluorescein isocyanate. Results indicate a reaction of substances within the optic vesicle with the fluorescein conjugated lens antisera prior to lens induction. 6. The inhibition of the lens-inducing capacity of the optic vesicle by the anti-adult lens sera suggests that substances within the optic vesicle, which can react with adult lens antibodies, are essential in the inductive process. We are indebted to Dr. William E. Dossel experiments. We wish to thank Dr. Edward allowing us to use his fluorescent microscope.
for his assistance C. Horn, Duke
in the in University,
vitro for
REFERENCES
ALEXANIIEH, L. E. (1937). overlying 41-73.
ectoderm
in lens
.4n experimental study of the role of optic formation in the chick embryo. J. Exptl.
cup Zool.
and 75,
COONS, A. H., cells. II. fluorescent
DAVIDSON, 23, 33. DOSSEL, W.
and KAPLAN, M. H. (1950). Localization of antigen in tissue Improvement in a method for the detection of antigen by means of antibody. J. Exptl. Med. 91, l-13. M. H. (1945). The preparation of frog embryo slides. Ttcrtox Neto.v
E. ( 1958). Preparation of tungsten micro-needles for use in cmresearch. Lab. Inuest. 7, 171-178. B., and ROBISON, R. ( 1929). The growth, development and phosactivity of embryonic avian femora and limb-buds cultivated in uitro. J. 23, 767-784. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. LANGMAN, J. ( 1956). Certain morphological aspects in the development of the crystalline lens in the chick embryo. Acta Morphol. Neerl. Stand. 1, 81-92. LAN~MAN, J., SCHALEKAMP, M. A. D. H., KUYKEN, M. P. A., aqd VEEN, R. ( 1957). Sero-immunological aspects of lens development in chick embryos. Acta Morphol. Neerl. Stand. 2, 142-154. LANNI, F., DILLON, M. L., and BEARD, J. W. (1950). Determination of small quantities of nitrogen in serological precipitates and other biological materials. J. Exptl. BioZ. and Med. 74, 4-7. LASH, J., HOLTZER, S., and HOLTZER, H. (1957). An experimental analysis of the development of the spinal column. VI. Aspects of cartilage induction. Exptl. Cell Research 13, 292-303. MCKEEHAN, M. S. ( 1951). Cytological aspects of embryonic lens induction in the chick. J. Exytl. Zool. 117, 31-64. RI~KEEHAN, M. S. (1958). Inductions of portions of the chick lens without contact with the optic cup. Anat. Record 132, 297-306. Nru. hf. C. (1956). New approaches to the problem of embryonic induction. In “Cellular Mechanisms in Differentiation and Growth” (I). Rudnick, ed. ), Chapter VII, pp. 155-171. Princeton Univ. Press, Princeton, New Jersey. bryologic FELL, H. phatase Biochem.
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AND
IRA
FOWLER
SIRLIN, J. L., and BRAHMA, S. K. ( 1959). Studies on embryonic induction using radioactive tracers. II. The mobilization of protein components during induction of the lens. Develop. Biol. 1, 234-246. SZEPSENWOL, J. ( 1933). Recherches sur les centres organisateurs des vksicules auditives chez des embryons de poulets omphalocephales obtenus experimentalement. Arch. anut. microscop. 29, 5-94. TEN GATE, G., and VAN DOORENMAALEN, W. J. ( 1950). Analysis of the development of the eye-lens in chicken and frog embryos by means of the precipitin reaction. Koninkl. Ned. Akad. Wetenschap. PTOC. 53, 894. VAN DETH, J. H. M. G. (1940). Induction et regeneration du cristallin chez l’embryo de la Poule. Acta Need. Morphol. 3, 219-236. VAN DOORENMAALEN, W. J. ( 1958). Histo-serological demonstration of the localization of lens-antigens in the embryonic chick-lens. Acta Morphol. Need. Scud 2, 1-12. WADDINGTON, C. H. (1937). The determination of the auditory placode in the chick. J. Exptl. Biol. 14, 232-239. WEISS, P. ( 1950). Perspectives in the field of morphogenesis. Quart. Rev. Biol. 25, 177-198. WOERDEMAN, M. W. (1955). Immunobiological approach to some problems of induction and differentiation. In “Biological Specificity and Growth” (E. G. Butler, ed.), pp. 33-53. Princeton Univ. Press, Princeton, New Jersey.
The
BIOLOGY,
2, 155-172
(
1960)
Inhibition of Lens-inducing Optic Vesicle with Adult M.
WILLIAM Department
of
Anutomy,
CLARKE’
Capacity of the Lens Antisera
AND
IRA
University of North North Carolina
Accepted
Janzrmy
FOWLER” Curolina,
Chapel
tlill,
26, 1960
INTRODUCTION
Several investigators (Alexander, 1937; van Deth, 1940; McKeehan, 1951; Langman, 1956) have pointed out that in chick embryos the lens originates from head ectoderm in contact with, and under the inductive influence of, the optic vesicle. Induction occurs during the period (9-21 somites) the optic vesicle adheres closely to the presumptive lens ectoderm ( McKeehan, 1951). Prior to the 9-somite stage, the presumptive lens ectoderm can be readily separated from the optic vesicle. Separation of the two tissues by mechanical means is extremely difficult at lo-21 somites of age. After 21 somites of age, the lens ectoderm attains capacity of self-differentiation. The presence of lens antigens in the embryonic chick lens has been demonstrated by serological methods (see Woerdeman, 1955; van Doorenmaalen, 1958, for review of the literature). Using fluoresceinconjugated antilens sera, van Doorenmaalen (1958) was unable to demonstrate a clear-cut localization of lens antigens prior to 5 days of incubation. With the precipitin method, ten Cate and van Doorenmaalen (1950) f ound adult lens antigens in the extract of lens vesicles of chick embryos as early as 58 hours’ incubation. Langman et al. (1957) reported evidence of the presence of precursors of lens proteins in 5-17 somite stages. This observation was based upon the cytolysis of presumptive lens ectoderm exposed, in vitro, to antiadult lens sera. 1 Aided by a Medical Student Fellowship from the National Foundation. 2 This investigation was supported by a senior Research Fellowship (SF-93-C from the Public Health Service. L55
)
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On the basis of an experiment (described more fully in the discussion of this paper) Woerdeman (1955) suggests that precursors in the brain and optic vesicles of early embryos may be involved in lens induction by reacting with precursors in the presumptive lens ectoderm. In such a system, precursors from both sources would be essential to the inductive process. If the precursors in the optic vesicle are similar to adult lens antigens, it might be possible to bind these precursors with adult lens antisera and thus prevent their activity in the induction process. The purpose of this investigation was to study the effect of adult lens antisera upon the inducing capacity of the optic vesicle by exposing the isolated optic vesicle to the antisera and subsequently testing its capacity to induce a lens in competent ectoderm. METHODS
1. Preparation
of Antisera
Adult chicken lenses obtained from freshly killed chickens were dissected free of the lens capsule and homogenized with an equal volume of physiological saline in. an all-glass (ten Broeck) homogenizer at 0-4O C. The homogenate was centrifuged at 14,000 x gravity at O-2” C for 15 minutes in a Servall centrifuge. Total nitrogen determinations (method of Lanni et aZ., 1950) showed that the supernatant contained approximately 100 mg of protein per milliliter. Two New Zealand white rabbits were immunized with this supernatant, each receiving five injections of the antigen; the injections were given on alternate days over a lo-day period. Each rabbit received a total of 288 mg of protein. Fourteen days after the initial injection of the antigen, the sera were collected and pooled. The sera gave a precipitate with all dilutions of the antigens up to and including one containing 0.001 mg of protein per milliliter. The crude sera were fractionated by the ammonium sulfate technique; the precipitated globulin was resuspended in one-fourth of the original volume in physiological saline buffered at pH 7.0 and was then stored at O-40 C until used. The interface precipitin test was the same as indicated with original crude antisera. Normal rabbit sera were fractionated similarly for use in the controls. 2. Fluorescent Normal
rabbit
Procedures globulin
and antilens globulin
were
conjugated
with
ANTISERA
ACTION
IN
LENS
157
INDUCTION
fluorescein isocyanate (Sylvana Chemical Company) according to the technique of Coons and Kaplan (1950). The fluorescent conjugate was employed as a histochemical stain for lens antigens in sections of embryos of 7 somites to 5 days of age. The yellow-green fluorescence on a black background marked the site of antigen localization in the section. Embryos were prepared according to the technique of Davidson ( 1945). Following rehydration one drop of the fluorescent globulin was placed on the section; after this it was incubated in a humidified chamber at 37O C for 30 minutes. Excess globulin was removed by washing in diethyl barbiturate buffer at pH 8.1 for 20 minutes. Sections were mounted in a mixture of buffered saline and glycerol and examined under the fluorescent microscope (Leitz ortholux microscope with ultraviolet illuminator). Photomicrographs were prepared using plus X film. Sections adjacent to the one stained with fluorescent antilens sera were employed as controls. These included: (1) a section stained with fluorescent normal rabbit globulin; (2) a section treated with unconjugated antilens globulin followed by the fluorescent antilens globulin; and (3) one unstained section.
#199+ 10% horse sera E= ‘120 anti-adult lens 6ero C= kzo normal rabbit ?,er(l I8 hours
%I99
t 10% horse
sere
FIG.
Procedure
1.
3. Effect of Antilens
Earle’s solutmn 30 minutes
7-somite
used
in
in vitro
studies
(see
text
for
ectoderm
details).
Sera in Vitro
The operative procedure is outlined in Fig. 1. Optic vesicles of embryos of P7 somites (Hamburger and Hamilton, 1951, stages 8 and 9) were removed, free of ectoderm, with tungsten needles prepared according to Dossel (1958). Care was taken to include a minimum of brain with the optic vesicle, although mesenchyme was not en-
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tirely removed. Each optic vesicle was transferred to a glass microculture slide containing 19 parts artificial medium 199 (Microbiological Associates) with 10% horse serum added and 1 part of adult-lens antisera (globulin fraction containing 0.001 mg of protein per milliliter). The vesicle was allowed to settle to the bottom of the concavity. TWO microculture slides were placed in a petri dish on moistened filter paper (a modification of the culture technique of Fell and Robison, 1929) and -incubated for a period of 18 hours at 38’ C. After 18 hours of culture, the optic vesicles were removed from the culture medium and washed for 30 minutes in at least three changes of Earle’s solution to remove as much of the lens antisera as possible. The vesicles were then returned to individual culture slides and incubated until strips of ectoderm could be prepared for the next phase of the experiment. Relatively large strips of ectoderm were removed from embryos of 4-7 somites that had been stained lightly with Nile blue sulfate. The ectoderm removed included the presumptive lens ectoderm anteriorly, and extended posteriorly to approximately the future level of the otocyst. Laterally, it included all the embryonic area. The medial incision was immediately lateral to the brain, The piece of ectoderm was washed in Earle’s solution and transferred immediately to a culture dish containing an optic vesicle. The optic vesicle, during the B-hour culture period, typically rounded up into a ball, which facilitated its enclosure in the ectoderm. The ectoderm strip was spread out on the bottom of the glass dish with surface side down, and the round optic vesicle was moved on top of the ectoderm. The ectoderm and vesicle were rolled in such a way as to wrap the vesicle completely within the ectoderm. The ectoderm frequently was overlapped. A glass needle was run through the combined tissues to attempt to ensure direct contact between the two layers (see Fig. 1). The needle was removed the following day (approximately 12 hours later ) . The combined optic vesicle-ectoderm cultures were incubated for a period of 3-4 days. The cultures were then fixed in Rouin’s solution, sectioned at 7 p., and stained with hematoxylin. The same experimental procedure was followed in the controls, using the globulin fraction of normal rabbit sera instead of adult-lens antisera during the 18-hour culture period prior to enclosing the optic vesicle in ectoderm. Optic vesicles with the overlying ectoderm intact were cultured in
ANTISERA
ACTION
IN
LENS
INDUCTION
159
19 parts medium 199 + 10% horse serum and 1 part adult lens antisera in procedure similar to that used by Langman et al. (1957) to determine if the antisera would prevent the formation of a lens at the concentration used in these experiments. No lenses formed in the presence of the antisera. As an additional control, sections of the hindbrain and overlying ectoderm, which included the presumptive otocyst, were grown in the above medium containing a 120 dilution of the antisera to determine whether or not the antisera used were exerting a specific effect upon differentiation of the lens or a generalized effect that would prevent differentiation of the otic vesicle. 4. In Vivo Studies
Optic vesicles were removed free of ectoderm from 4-7 somite embryos and cultured for 18 hours as in the in vitro studies previously described. In the experimental series the liquid medium contained a 1:20 dilution of anti-adult lens globulin; in the control series a similar dilution of normal rabbit globulin was used. At the end of the 1% hour culture period the vesicles were washed in Earle’s solution and implanted in host embryos P7 somites of age. The implant site was immediately posterior to the host optic vesicle. Care was taken to locate the vesicle between the ectoderm and underlying mesoderm so that there would be little or no mesoderm between the vesicle and ectoderm. After the implant was in place, the eggs were sealed and incubated for an additional period of 3-4 days. The embryos were fixed in Bouin’s solution, and the region of the graft was sectioned serially and stained in hematoxylin. RESULTS
Optic vesicles extirpated, free of ectoderm, from embryos of 4-7 somites and cultured on glass in liquid medium evidenced little growth during the initia1 H-hour culture period. The cut edges of the vesicle tended to heal together, so that after 18 hours, the vesicle was round. Thus, when the vesicle was wrapped in ectoderm, the sheet of ectoderm was in direct contact with the presumptive retina (the part normally in contact with presumptive lens ectoderm). Len9
lntl~rc+on
in Vitro
Treatment of the optic vesicles for a period of 18 hours with adult
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lens antisera had no detectable morphological effect in comparison with vesicles similarly cultured in normal rabbit sera. Some cytolysis occurred in both the experimental series in which the optic vesicles were exposed to the adult lens antisera (Figs. 2-4, Plate I) and in the
PLATE I C&l. The optic vesicles were treated with adult-lens antisera for 18 prior to enclosure in ectoderm. FIG. 5. The optic vesicle was treated with rabbit sera for 18 hours prior to enclosure in ectoderm. FIGS. 2-5. Exwere cultured 4 days after enclosure in ectoderm. Magnification: X 100. 2. The ectoderm was lost during the culture period. The optic vesicle increased in size, but there was no evidence of “cup” formation. 3. The optic vesicle (0.0.) maintained close contact with the ectoderm but no lens formed. A lens formed in only 2 of 34 similar explants. 4. The optic vesicle ( o.u.) is widely separated from the ectoderm No lens formed in this type of explant. 5. The lens is well developed, although there is considerable necrosis the optic vesicle. A lens differentiated in 35 of 43 explants of this type.
FIGS.
hours normal plants FIG. (0.0.) FIG.
(ect.), FIG.
(ect.). FIG.
within
ANTISERA
ACTION
IN
LENS
161
INDUCTION
control series, in which the optic vesicles were exposed to normal rabbit sera (Fig. 5). Cytolysis within the optic vesicle did not appear to affect directly its capacity to induce a lens. Cytolysis within the optic vesicle was, perhaps, a factor in preventing maintenance of contact between the ectoderm and the optic vesicle. The most striking difference between the two series was in the differentiation of the ectoderm in response to contact with the optic vesicles. Results of both the control series and the experimental series are summarized in Table 1. In 14 of the controls and 22 experimentals, the ectoderm
LENS
Denuded Ectoderm Ectoderm Total
DEVELOPMENT
TABLE 1 IN HEAI) ECTODERM WITH OPTIC VESICLES
vesicle not fused with O.V. fused with O.V.
14 9 43 66
0 0 35 35
IN RESPOXSE in Vitro
0 0 81 53
22 5 34 61
TO CONTACT
0 0 2 2
0 0 6 3
a Prior to enclosure in head ectoderm the optic vesicles (o.v.) were cultured 18 hours in a 1: 20 dilution of normal rabhit serum in Medium 199 plus 10% horse sera. b Prior to enclosure in head ectoderm, the optic vesicles were cultured 18 hours in a 1:20 dilution of the anti-adult lens sera in Medium 199 plus 10% horse sera.
was lost in the process of handling the vesicles. These cultures (see Fig. 2) were the result of experimental error. Since there was no opportunity for lens induction to occur in these cases, they furnish no information as to the lens-inducing capacity of these vesicles. In 9 of the controls and 5 of the experimental cultures, the ectoderm enclosed, but did not maintain direct contact with, the optic vesicles. An example of this type of culture is shown in Fig. 4. The ectoderm is separated from the optic vesicle by considerable space, which was presumably filled with culture medium. Induction of a lens does not occur under these conditions ( McKeehan, 1951; Langman, 1956), although McKeehan (1958) has been able to obtain lenses in viva without direct contact between optic vesicle and presumptive lens ectoderm. Since the above cases (denuded vesicles and cultures in
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ANTISERA
ACTION
IN
LENS
INDUCTION
163
which ectoderm did not fuse with the optic vesicle) failed to provide opportunity for lens induction to occur, they are eliminated from consideration in the evaluation of the data regarding the effect of the anti-adult lens sera upon the lens-inducing capacity of the optic vesicle. The remaining cultures (43 of the control series and 34 of the experimental series) are useful in assessing the lens-inducing capacity, since in these cases the ectoderm established and maintained contact with the optic vesicle (see Table 1). In 35 of 43 cultures in which the optic vesicles were exposed 18 hours to a 1:20 dilution of normal rabbit sera prior to enclosure in competent ectoderm, a lens was induced in ectoderm (Fig. 5). This is contrasted with the cultures in which the optic vesicles were treated with a 1:20 dilution of antiadult lens sera prior to enclosure in competent head ectoderm. In these cases, illustrated in Fig. 3, a lens developed in only 2 of 34 (6%) cultures, even though extensive area of contact between ectoderm and vesicle was demonstrated histologically. The lenses produced were in the form of placodes similar to that shown in Fig. 8 ( Plate II ) . In cultures in which the ectoderm was overlapped extensively, as is shown in Fiq. 6. the optic vesicles failed to grow and excessive cytolysis occurred in both the control and experimental series. Apparently, exchange between the cells of the optic vesicle and surrounding
FIGS.
FIG. dcrm necrotic, cultured FIG.
(h.h.)
PLATE II 6-9. Magnification: x 100. 6. The optic vesicle is completely enclosed in a double layer of ectoas a result of extensive overlan of the ectoderm. The optic vesicle is although it is a control vesicle treated with normal rabbit sera. Explant 3 days after the optic vesicle was enclosed in ectoderm. 7. Explant included a lateral half of the primordium of the hindbrain and the adjacent ectoderm. An otic vesicle (ot.u.) developed in the
ectodcrm. FIG. 8. The lens is in the form of a placode and is separated from the optic vesicle (0.0.). Presumably, the lens ectoderm was in contact with the optic vesicle during induction. The optic vesicle was a control vesicle exposed to normal rabbit sera and cultured 4 days after enclosure in ectoderm. FIG. 9. Exulant included the entire primordium of the hindbrain and the adjacent ectoderm. An otic vesiclr (ot.0.) developed in the ectoderm. The nervous tissue is completely enclosed in ectoderm and contains more necrotic cells than does the nervous tissue (lateral half of hindbrain) shown in Fig. 7.
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PLATE III FIGS. 10-12. Magnification: x 50. FIG. 10. The donor optic vesicle (d.o.o.) was exposed to normal rabbit sera for 18 hours prior to implantation in the host embryo. A lens (d.2.) was induced in the host ectoderm. This lens is smaller than the lens (h.Z.) of the host embryo. FIG. 11. Procedure was similar to that followed in specimen shown in Fig. 10. The lens (d.l.) induced in the host embryo by the donor optic vesicle (d.o.u.) is comparable in size and differentiation to that of the host lens (h.l.).
ANTISERA
medium was insufficient of ectoderm. Lens Induction
ACTION
IN
to maintain
LENS
165
INDUCTION
cells enclosed in a double
layer
in Vivo
By implanting optic vesicles in host embryos, an attempt was made to determine whether the failure of the optic vesicles exposed to antiadult lens sera in vitro was, perhaps, due to relatively poor conditions for lens differentiation. Mortality of the host embryos was high, and the optic vesicle slipped out of the implantation site in a number of specimens. In 3 of 5 specimens in which control vesicles were implanted, contact between donor optic vesicle and host ectoderm was demonstrated histologically. In each of these 3 cases a lens developed from the host ectoderm (see Figs. 10 and 11, Plate III). In 5 of 7 specimens in which experimental vesicles (previously exposed to anti-adult lens sera) were implanted, contact between donor optic vesicle and host ectoderm was observed as illustrated in Fig. 12 (Plate III). No lens differentiated in the host ectoderm in response to the influence of these optic vesicles; these results conform to the findings in the in vitro studies. Effect of Anti-adult
Lens Sera upon Formation of Otic Vesicle
To test whether the antilens sera would have a generalized effect upon induction of other head structures, its capacity to block the formation of the otic vesicle was studied. The otic vesicle develops from head ectoderm under the inductive influence of the hindbrain (Waddington, 1937) and adjacent neural crest (Szepsenwol, 1933). Induction is probably complete by about 4 somites of age. Primordia of hindbrain (either whole or a lateral half) together with the overlying ectoderm from embryos younger than 4 somites were cultured in a medium containing a 1:20 dilution of the anti-adult lens sera. In each of 16 cases (see Figs. 7 and 9) the otic vesicle was formed even in cultures in which the hindbrain tended to cytolyze. Thus it is evident that the antisera did not prevent induction of the otic FIG. 12. The sera for 18 hours the host ectoderm host ectoderm.
donor optic vesicle (d.o.u.) was exposed prior to implantation in the host embryo. even though the optic vesicle established
to anti-adult lens No lens formed in contact with the
ANTISERA
ACTIOK
IN
LENS
INDUCTION
vesicle at the concentration used in this study. The action appears be specific in its effect upon eye structures. Normal
Embryos
Stained
with
Fluorescent
167 to
Antisera
In an attempt to determine why there was apparently a specific effect of the antilens sera upon the lens-inducing capacity of the optic vesicles, a series of normal embryos from 7 somites to 5 days of age were stained with anti-adult lens sera conjugated with fluorescein isocyanate. Sections of selected stages stained with the fluorescent antisera are illustrated in Figs. 13-16 (Plate IV). These sections were stained with hemotoxylin following the fluorescent study and are shown in Figs. 17-20 (Plate IV). In normal S-day embryos, the lens shows an intense fluorescence following treatment with the fluorescent antisera. The results at this age appear to be similar to those of van Doorenmaalen ( 1958). In younger embryos, van Doorenmaalen found only a generalized nonspecific type staining. Owing, perhaps, to the difference in the fixative used (van Doorenmaalen used acetone; we used Smith’s Fluid), the younger embryos appeared to stain more intensely than was the case in van Doorenmaalen’s careful study. As may be seen in comparing Figs. 13, 14, 15, and 16 the intensity of staining is progressively less in the younger embryos. In embryos of I9 somites (Fig. 14)) at which induction is almost complete, the optic vesicle and presumptive lens ectoderm fluoresce with approximately c~rual intensity. In embryos of 12 somites (Fig. 13), prior to lens PLATE IV FIGS. 13-16. Normal embryos stained with anti-adult lens sera conjugated with fluorescein isocyanate. Microphotographs were made with uniform conditions of lighting, exposure time, and magnification. The degree of lightness in the print reflects the intensity of fluorescence of the specimen. Magnification: x 100. FIG. 13. Embryo of 12 somites. The optic vesicle (0.0.) fluoresces with greater intensity than does the adjacent ectoderm (ect. ) . Embryo of 19 somites. The optic vesicle and adjacent lens ectoderm FIG. 14. fluoresce with approximately equal intensity. The lens of a 4-day embryo. The intensity of fluorescence is greater FIG. 15. than in Figs. 13 and 14, but less than is seen in Fig. 16. FIG. 16. The lens of a 5-day embryo. Fluorescence is localized in the lens and is of greater intensity than in younger stages. FIGS. 17-20. Same as Figs. 13-16; stained with hematoxylin. Magnification: x 100.
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induction, the optic vesicle fluoresces with greater intensity than does the presumptive lens ectoderm. This indicates that there are substances in the optic vesicle prior to lens induction that react with fluorescein conjugated adult lens antisera. The reaction of substances within the presumptive lens ectoderm with the fluorescent lens antisera increases as lens induction proceeds; simultaneously, the reaction of substances within the optic cup with fluorescent lens antisera decreases so that by 5 days this reaction is, as van Doorenmaalen has shown, localized within the lens. DISCUSSION
It is well known that inducing and induced tissues have an intimate association with one another. This is especially well demonstrated in the development of the lens, where certain cytological changes in the presumptive lens ectoderm are shown to occur in response to contact with the optic vesicle. McKeehan (1951) has described in detail the cytological changes in the presumptive lens epithelium, consisting of loss of vacuolization, nuclear orientation, and palisade formation. Weiss (1956) suggests the nuclear orientation may indicate simultaneous molecular orientation at the cell surface. McKeehan (1958) has observed a few instances of lenses induced in ectoderm separated from the optic vesicle by a thin agar membrane. This suggests substances of a diffusible nature may be involved in the lens induction process. Evidence is accumulating that inductors in general are diffusible. The experiments of Lash et al. (1957) demonstrate that neural tube substances can penetrate a porous membrane and cause differentiation of cartilage, although this process may not be induction in the classic sense. The studies of Niu (1956) indicate clearly that the neural inductor with which he is working is diffusible. The appearance of specific antigenic substances has been shown in many instances, to be correlated with morphological events. However, there is little evidence that specific antigens play a causal role in differentiation. Woerdeman (1955) suggests such a role in lens development. Saline extracts of head ectoderm of young axolotl neurulae before the appearance of a lens placode failed to react with antisera to adult lens. The same was found to be true with extracts of young, isolated eye vesicles. The extracts of eve vesicle and presumptive lens ectoderm were mixed and incubated at 370 C
ANTISERA
ACTION
IN
LENS
INDUCTION
169
for 24 hours. This mixture then showed a precipitin reaction with lens antisera. Presumably, new compounds were formed in vitro that reacted with lens antisera. From this it might be inferred that the production of lens antigens results from the interaction between head ectoderm and eye vesicle. Such a system would explain the results of our experiments. If the eye vesicle substances involved in the above process contained groups that react with adult lens antisera, then these substances would be inactivated by reaction with antibodies to lens antigens and induction would fail. In our investigation, the staining of normal embryos with fluorescein-conjugated antisera indicates that there are indeed substances present in the optic vesicle prior to and during the induction process which can react with adult lens antisera. The inhibition of the lens-inducing capacity of the eye vesicle by adult-lens antisera suggests that these substances which react with the antisera are essential to the induction process. The nature of the substances is unknown. Sirlin and Brahma, 1959, have presented evidence of transfer of both small molecules and complex, more specific, molecules or particles from the optic vesicle to the lens during lens induction in Xenopus. Eyecups incubated with C4labeled phenylalanine and subsequently enclosed in ectoderm, in a procedure similar to that used in this investigation, induced lenses in the ectoderm. The tracer was clearly detectable in the developing lens. Eyecup cytoplasm near the lens was depleted of the labeled proteins, whereas labeling of the lens cytoplasm increased. Although this study does not show directly that the labeled proteins are involved in the induction of the lens, the shift of tracer into the lens during the period of induction is certainly suggestive. In our study, the molecules involved are probably not identical with lens proteins. They may merely happen to contain determinant groups capable of reacting with some part of one or more of the antibody molecules. On the other hand, the reaction in the optic vesicle that is evident from the staining of normal embryos with the fluorescein conjugated antibody may be a reaction between the antibody and compounds not involved in lens induction. This appears to be unlikely in view of the fact that treating the optic vesicles with unconiugated antibody virtually eliminated its capacity to induce a lens. The possibility that the action of the antilens sera is a generalized toxic effect rather than a specific reaction with certain compounds within the optic vesicle has not been eliminated conclusively in
170
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these experiments. However, the antisera did not prevent induction of the otocyst at the same concentration used in treating the optic vesicles. This, together with the fact that there was no detectable morphological effect of the antisera upon the optic vesicles in comparison with the controls, suggests a high degree of specificity in the action of the antisera. Langman et al. (1957) observed unmistakable evidence of an effect of their antilens sera upon the optic vesicle. At concentrations of antilens sera which were considerably higher than that used in this study, several of their preparations showed slight degeneration of the optic cup. The concentration of antisera used in our experiments produced no morphological changes that could be ascribed to the antisera. This was perhaps due to the slow growth medium used (artificial medium 199 with 10% horse sera added). Langman used embryonic extract. In the course of our investigation, cultures of optic vesicles with the overlying ectoderm left in place were exposed in artificial medium 199 to a 1:20 dilution of antilens sera. Typical lenses failed to develop in 100% of the cases. In similar cultures with normal rabbit sera substituted for the antilens sera, lenses developed in 75% of the cultures. The inhibitory action of antilens sera appears to be more effective when used in artificial medium than when added to embryonic extract. SUMMARY
1. Optic vesicles removed, free of ectoderm, from chick embryos of 4-7 somites were explanted for 18 hours in a medium containing a 1:20 dilution of the globulin fraction of anti-adult lens sera. The vesicles were then enclosed in competent ectoderm and cultured 3-4 days in a medium free of the antisera. Induction of a lens occurred in 2 of 61 cases (3%). 2. Induction of a lens resulted in 35 of 66 cases (53%) in the control vesicles cultured in a 1:20 dilution of normal rabbit sera (globulin fraction) and subsequently enclosed in ectoderm. 3. The results of experiments in which optic vesicles were cultured in media containing either antilens sera or normal rabbit sera and subsequently implanted beneath the ectoderm of 4-7 somite embryos conform to the results of the in vitro experiments. 4. A 1:20 dilution of the anti-adult lens sera failed to inhibit development of the otic vesicle. 5. Sections of selected stages of normal embryos were stained
ANTISERA
ACTION
IN
LENS
171
INDUCTION
with antilens sera conjugated with fluorescein isocyanate. Results indicate a reaction of substances within the optic vesicle with the fluorescein conjugated lens antisera prior to lens induction. 6. The inhibition of the lens-inducing capacity of the optic vesicle by the anti-adult lens sera suggests that substances within the optic vesicle, which can react with adult lens antibodies, are essential in the inductive process. We are indebted to Dr. William E. Dossel experiments. We wish to thank Dr. Edward allowing us to use his fluorescent microscope.
for his assistance C. Horn, Duke
in the in University,
vitro for
REFERENCES
ALEXANIIEH, L. E. (1937). overlying 41-73.
ectoderm
in lens
.4n experimental study of the role of optic formation in the chick embryo. J. Exptl.
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COONS, A. H., cells. II. fluorescent
DAVIDSON, 23, 33. DOSSEL, W.
and KAPLAN, M. H. (1950). Localization of antigen in tissue Improvement in a method for the detection of antigen by means of antibody. J. Exptl. Med. 91, l-13. M. H. (1945). The preparation of frog embryo slides. Ttcrtox Neto.v
E. ( 1958). Preparation of tungsten micro-needles for use in cmresearch. Lab. Inuest. 7, 171-178. B., and ROBISON, R. ( 1929). The growth, development and phosactivity of embryonic avian femora and limb-buds cultivated in uitro. J. 23, 767-784. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. LANGMAN, J. ( 1956). Certain morphological aspects in the development of the crystalline lens in the chick embryo. Acta Morphol. Neerl. Stand. 1, 81-92. LAN~MAN, J., SCHALEKAMP, M. A. D. H., KUYKEN, M. P. A., aqd VEEN, R. ( 1957). Sero-immunological aspects of lens development in chick embryos. Acta Morphol. Neerl. Stand. 2, 142-154. LANNI, F., DILLON, M. L., and BEARD, J. W. (1950). Determination of small quantities of nitrogen in serological precipitates and other biological materials. J. Exptl. BioZ. and Med. 74, 4-7. LASH, J., HOLTZER, S., and HOLTZER, H. (1957). An experimental analysis of the development of the spinal column. VI. Aspects of cartilage induction. Exptl. Cell Research 13, 292-303. MCKEEHAN, M. S. ( 1951). Cytological aspects of embryonic lens induction in the chick. J. Exytl. Zool. 117, 31-64. RI~KEEHAN, M. S. (1958). Inductions of portions of the chick lens without contact with the optic cup. Anat. Record 132, 297-306. Nru. hf. C. (1956). New approaches to the problem of embryonic induction. In “Cellular Mechanisms in Differentiation and Growth” (I). Rudnick, ed. ), Chapter VII, pp. 155-171. Princeton Univ. Press, Princeton, New Jersey. bryologic FELL, H. phatase Biochem.
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SIRLIN, J. L., and BRAHMA, S. K. ( 1959). Studies on embryonic induction using radioactive tracers. II. The mobilization of protein components during induction of the lens. Develop. Biol. 1, 234-246. SZEPSENWOL, J. ( 1933). Recherches sur les centres organisateurs des vksicules auditives chez des embryons de poulets omphalocephales obtenus experimentalement. Arch. anut. microscop. 29, 5-94. TEN GATE, G., and VAN DOORENMAALEN, W. J. ( 1950). Analysis of the development of the eye-lens in chicken and frog embryos by means of the precipitin reaction. Koninkl. Ned. Akad. Wetenschap. PTOC. 53, 894. VAN DETH, J. H. M. G. (1940). Induction et regeneration du cristallin chez l’embryo de la Poule. Acta Need. Morphol. 3, 219-236. VAN DOORENMAALEN, W. J. ( 1958). Histo-serological demonstration of the localization of lens-antigens in the embryonic chick-lens. Acta Morphol. Need. Scud 2, 1-12. WADDINGTON, C. H. (1937). The determination of the auditory placode in the chick. J. Exptl. Biol. 14, 232-239. WEISS, P. ( 1950). Perspectives in the field of morphogenesis. Quart. Rev. Biol. 25, 177-198. WOERDEMAN, M. W. (1955). Immunobiological approach to some problems of induction and differentiation. In “Biological Specificity and Growth” (E. G. Butler, ed.), pp. 33-53. Princeton Univ. Press, Princeton, New Jersey.