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Neuro-retinal “lentoids” contain no lens antigens
Experimental Cell Research 66 (1971) 65-68
NEURO-RETINAL
"LENTOIDS"
CONTAIN
NO LENS ANTIGENS
Immunofluorescent Criteria A. KATOH, 1 M. BRAVERMAN2 and CHARLOTTE YEH 1 lOneology Laboratory of the Division of Radiotherapy, Mercy Hospital, Pittsburgh, Pa 15219, and 2Cellular and Radiation Biology Laboratories, Department of Radiology, Allegheny GeneralHospital, Pittsburg, Pa 15212, USA
SUMMARY "Lentoids" differentiating in cultures of dissociated and reaggregated chick 7-day embryo neural retain contain no antigens cross reacting, by immunofluorescent criteria, to total chicken lens antiserum.
In 1957, M o s c o n a [I] described the f o r m a t i o n of lens-like bodies which he called "lentoids" in dissociated and reaggregated chick neural retina cells. Earlier reports [2-5] indicated that dissociated cells of a variety of embryonic tissues reaggregate histotypically according to their origin. The formation of lens-like histology by neuro-retinal reaggregates constituted an exceptional instance where a histogenetic developmental pathway was rechannelled into an altered course. Braverman, C o h e n & K a t o h [6], however, were unable to demonstrate lens antigens in neuroretinal reaggregates analysed by immunodiffusion techniques. Paradoxically, they reported that when dissociated neuro-retinal cells were cultured during the reaggregation phase in the presence of antiserum to chick, beef, or sheep lens a cytotoxic reaction resuited. Other types of tissues reaggregated under the same culture conditions were unaffected. A l t h o u g h quantities of lens antigens sufficient to give a positive reaction in immunodiffusion are not present in lentoid5 - 711806
bearing reaggregates, the possibility remains that ceils of the lentoid are synthesizing lens protein. Conceivably the antigen p r o d u c e d in these cells might be sufficiently diluted in a soluble extract to escape detection. We n o w report the application of the indirect immunofluorescence m e t h o d for the detection of lens antigens in reaggregates of 6-7 day chick e m b r y o neuro-retinal tissue. MATERIALS AND METHODS Culture methods for the dissociation and reaggregation of 6-7 day chick embryo neuro-retinal cells are detailed in Braverman et al. [6]. For the immunofluorescence technique, tissues were fixed in ice-cold 95 % ethanol, and embedded in paraffin according to the method of Sainte-Marie [7]. Fluorescent goat globulin against rabbit gamma globulin was purchased from Microbiological Associates, Bethesda, Md. This material was absorbed with mouse powder (Difco) according to the protocol of Ikeda & Zwaan [8], and with a lyophilized preparation of headless 7 day chick embryos. Finally, the fluorescent globulin fraction was passed through a column of Sephadex C-25. Adult chicken lens antiserum was prepared in rabbits. An extract of adult chicken lenses in 0.15 M NaC1 was emulsified with an equal volume of Freund complete adjuvant (Difco) and 2.0 ml doses were Exptl Cell Res 66
66
A. Katoh et al.
Fig. 1. Immunoelectrophoresis of the immunizing antigen: adult chicken lens, versus the resulting antiserum. The three characteristic lens crystallin bands are shown, alpha, beta, and delta (F.I.S.C.). Fig. 2. Immunodiffusion (Ouchterlony) tests for the specificity of the lens antiserum were carried out against saline extracts of 7-day chick embryo (a) brain, (b) heart and (c) neural retina. Wells 1, 3, and 5 were filled with the tissue extracts; well no. 2 contained balanced salt solution; and well no. 4 contained chicken lens extract. Fig. 3. Section through a neuro-retinal reaggregate showing a lentoid in the upper left, rosette in the lower right. Hematoxylin and eosin staining. Bar measures 50 #. Fig. 4. High power view of the lentoid shown in fig. 3. Bar measures 50/~. Fig. 5. Immunofluorescence photograph of a 96 h embryo lens. Fluorescence is restricted to the lens. Bar measures 50/~. Fig. 6. Neuro-retinal reaggregate fixed and prepared for immunofluorescence, and photographed with phase contrast. A lentoid is shown in the center of the field. Bar measures 50/~. Fig. 7. The same section as that shown in fig. 6, but photographed with fluorescence optics. Lentoid exhibits no fluorescence. Lens antiserum used for figs 1, 2, 5, 6 is the same preparation.
Exptl Cell Res 66
Neuro-retinal "lentoids" injected subcutaneously at different sites over a period of 3 months. Antisera so prepared were tested for tissue specificity in agar immunodiffusion plates (Ouchterlony) and for the presence of alpha, beta, and delta (F.I.S.C.) crystallin bands by immunoelectrcphoresis. Immunoelectrophoresis was conducted on 1 x 3 inch microscope slides coated with 2.0 ml of 0.85 % ionagar no. 2 (Colab) in barbital buffer, pH 8.6. The electrophoresis was run at 150 volts for 90 min, and the pattern developed overnight with antisera in a moist chamber in the cold room (4~ Preimmunization sera from the same rabbits that supplied the lens antiserum was used as controls during the immunofluorescent technique. Tissue sections were cut at 3-4 /~, mounted on slides, and stained according to the protocol of Ikeda & Zwaan [8]. For fluorescence microscopy, a Leitz orthoplan microscope equipped with a high-pressure mercury lamp (HBO-200), exciter filters BG38 and UG 1, and barrier filters K430 and K460 were used with a dark field condenser. Photographs were taken on Kodak Tri-X 35 mm film.
RESULTS
Antiserum characterization W h e n tested against the i m m u n i z i n g antigen, the a n t i s e r u m d e m o n s t r a t e s three clean lines in i m m u n o e l e c t r o p h o r e s i s (fig. 1), a n d no cross r e a c t i o n to non-lens tissues can be visualized in O u c h t e r l o n y plates (fig. 2a-c). Lens antigens are u n a m b i g u o u s l y d e m o n s t r a t e d b y this s e r u m as b r i g h t fluorescence in the lens of a 96 h chick e m b r y o (fig. 5).
Lentoid analysis L e n t o i d s , identical to those illustrated b y M o s c o n a [1] f o r m with low frequency in n e u r a l retinal reaggregates. These are characterized, as are M o s c o n a ' s , b y lighter staining a n d e n l a r g e d cell nuclei, increased cytop l a s m a n d a t e n d e n c y for cells to b e c o m e p e a r s h a p e d (figs 3, 4). N e u r a l retinae f r o m 7-day old e m b r y o s were t r e a t e d in various ways, c u l t u r e d a n d analysed, utilizing imm u n o f l u o r e s c e n t techniques for lens antigens. N e i t h e r n e u r a l retina, n e u r a l retina dissociated, r e a g g r e g a t e d a n d cultured for 3 days, n o r n e u r a l r e t i n a dissociated, reaggregated, c u l t u r e d for 3 days a n d post-cultured, in the m a n n e r d e s c r i b e d b y M o s c o n a [1] gave a n y
67
i n d i c a t i o n of the presence of lens antigens (rigs 6, 7). DISCUSSION T h e structure described as " l e n s - l i k e " a n d " l e n t o i d " b y M o s c o n a , p r e s u m a b l y on the basis of its m o r p h o l o g y , does n o t in fact r e s e m b l e the a v i a n lens at a n y stage of its development. It resembles slightly a newt lens [9], b u t l o o k s m o s t like a k e r a t i n pearl [10]. These pearls are characteristically f o u n d in well differentiated e p i d e r m o i d carcinomas. The i m m u n o f l u o r e s c e n t studies r e p o r t e d here establish f u r t h e r that these p u t a t i v e lenslike structures are n o t synthesizing the u n i q u e a n d characteristic crystallins of the lens. W h a t d e v e l o p m e n t a l p a t h these structures are following is n o t clear; they are not, however, c a r r y i n g out the synthetic activities which characterize lens differentiation f r o m the fortieth h o u r on. The a p p e a r a n c e of these p s e u d o - l e n t o i d s in n e u r o - r e t i n a l reaggregate cultures treated with low c o n c e n t r a t i o n s of lens a n t i s e r u m [6], as well as the defects of the optic cup a n d b r a i n [11-15] o c c u r r i n g in e m b r y o s e x p o s e d to lens specific a n t i s e r u m continues to be p a r a d o x i c a l unless the p r e s u m p t i o n is m a d e t h a t this assay d e m o n s t r a t e s a n t i s e r u m c o m p o n e n t s t h a t are n o t visualized b y the sensitive i m m u n o l o g i c a l techniques b y which the sera were tested for cross r e a c t i o n to heterologous tissues. Technical assistants: Cindy Hambrick, Mona Tu, and Betty Ford. This investigation was supported by Atomic Energy Commission contract AT (30-1)-3825.
REFERENCES 1. 2. 3. 4. 5.
Moscona, A, Science 125 (1957) 598. - - Exptl cell res 3 (1952) 535. J anat 86 (1952) 287. - - Proc soc exptl biol med 92 (1956) 410. - - Proc natl acad sci US 43 (1957) 184. -
-
Exptl Cell Res 66
68
A. Katoh et al.
6. Braverman, M, Cohen, C & Katoh, A, J embryoI exptl morph 21 (1969) 391. 7. Sainte-Marie, G, J histochem cytochem 10 (1962) 250. 8. Ikeda, A & Zwaan, J, Invest ophthalmol 5 (1966) 402. 9. Reyer, R, J exptl zool 139 (1958) 137. 10. Alien, A, The skin, a clinical pathological treatise, p. 894. Grune & Stratton, New York (1966). 11. Burke, V J, Sullivan, N P, Petersen, H & Weed, R, J infect dis 74 (1944) 225.
Exptl Cell Res 66
12. Langman, J, Anat rec 137 (1960) 135. 13. Fowler, I & Clarke, W M, Anat rec 136 (1960) 194. 14. Langman, J & Maisel, H, Invest ophthalmol 1 (1962) 396. 15. Langman, J, Maisel, H & Squires, J, J embryol exptl rnorph 10 (1962) 178.
Received November 17, 1930
NEURO-RETINAL
"LENTOIDS"
CONTAIN
NO LENS ANTIGENS
Immunofluorescent Criteria A. KATOH, 1 M. BRAVERMAN2 and CHARLOTTE YEH 1 lOneology Laboratory of the Division of Radiotherapy, Mercy Hospital, Pittsburgh, Pa 15219, and 2Cellular and Radiation Biology Laboratories, Department of Radiology, Allegheny GeneralHospital, Pittsburg, Pa 15212, USA
SUMMARY "Lentoids" differentiating in cultures of dissociated and reaggregated chick 7-day embryo neural retain contain no antigens cross reacting, by immunofluorescent criteria, to total chicken lens antiserum.
In 1957, M o s c o n a [I] described the f o r m a t i o n of lens-like bodies which he called "lentoids" in dissociated and reaggregated chick neural retina cells. Earlier reports [2-5] indicated that dissociated cells of a variety of embryonic tissues reaggregate histotypically according to their origin. The formation of lens-like histology by neuro-retinal reaggregates constituted an exceptional instance where a histogenetic developmental pathway was rechannelled into an altered course. Braverman, C o h e n & K a t o h [6], however, were unable to demonstrate lens antigens in neuroretinal reaggregates analysed by immunodiffusion techniques. Paradoxically, they reported that when dissociated neuro-retinal cells were cultured during the reaggregation phase in the presence of antiserum to chick, beef, or sheep lens a cytotoxic reaction resuited. Other types of tissues reaggregated under the same culture conditions were unaffected. A l t h o u g h quantities of lens antigens sufficient to give a positive reaction in immunodiffusion are not present in lentoid5 - 711806
bearing reaggregates, the possibility remains that ceils of the lentoid are synthesizing lens protein. Conceivably the antigen p r o d u c e d in these cells might be sufficiently diluted in a soluble extract to escape detection. We n o w report the application of the indirect immunofluorescence m e t h o d for the detection of lens antigens in reaggregates of 6-7 day chick e m b r y o neuro-retinal tissue. MATERIALS AND METHODS Culture methods for the dissociation and reaggregation of 6-7 day chick embryo neuro-retinal cells are detailed in Braverman et al. [6]. For the immunofluorescence technique, tissues were fixed in ice-cold 95 % ethanol, and embedded in paraffin according to the method of Sainte-Marie [7]. Fluorescent goat globulin against rabbit gamma globulin was purchased from Microbiological Associates, Bethesda, Md. This material was absorbed with mouse powder (Difco) according to the protocol of Ikeda & Zwaan [8], and with a lyophilized preparation of headless 7 day chick embryos. Finally, the fluorescent globulin fraction was passed through a column of Sephadex C-25. Adult chicken lens antiserum was prepared in rabbits. An extract of adult chicken lenses in 0.15 M NaC1 was emulsified with an equal volume of Freund complete adjuvant (Difco) and 2.0 ml doses were Exptl Cell Res 66
66
A. Katoh et al.
Fig. 1. Immunoelectrophoresis of the immunizing antigen: adult chicken lens, versus the resulting antiserum. The three characteristic lens crystallin bands are shown, alpha, beta, and delta (F.I.S.C.). Fig. 2. Immunodiffusion (Ouchterlony) tests for the specificity of the lens antiserum were carried out against saline extracts of 7-day chick embryo (a) brain, (b) heart and (c) neural retina. Wells 1, 3, and 5 were filled with the tissue extracts; well no. 2 contained balanced salt solution; and well no. 4 contained chicken lens extract. Fig. 3. Section through a neuro-retinal reaggregate showing a lentoid in the upper left, rosette in the lower right. Hematoxylin and eosin staining. Bar measures 50 #. Fig. 4. High power view of the lentoid shown in fig. 3. Bar measures 50/~. Fig. 5. Immunofluorescence photograph of a 96 h embryo lens. Fluorescence is restricted to the lens. Bar measures 50/~. Fig. 6. Neuro-retinal reaggregate fixed and prepared for immunofluorescence, and photographed with phase contrast. A lentoid is shown in the center of the field. Bar measures 50/~. Fig. 7. The same section as that shown in fig. 6, but photographed with fluorescence optics. Lentoid exhibits no fluorescence. Lens antiserum used for figs 1, 2, 5, 6 is the same preparation.
Exptl Cell Res 66
Neuro-retinal "lentoids" injected subcutaneously at different sites over a period of 3 months. Antisera so prepared were tested for tissue specificity in agar immunodiffusion plates (Ouchterlony) and for the presence of alpha, beta, and delta (F.I.S.C.) crystallin bands by immunoelectrcphoresis. Immunoelectrophoresis was conducted on 1 x 3 inch microscope slides coated with 2.0 ml of 0.85 % ionagar no. 2 (Colab) in barbital buffer, pH 8.6. The electrophoresis was run at 150 volts for 90 min, and the pattern developed overnight with antisera in a moist chamber in the cold room (4~ Preimmunization sera from the same rabbits that supplied the lens antiserum was used as controls during the immunofluorescent technique. Tissue sections were cut at 3-4 /~, mounted on slides, and stained according to the protocol of Ikeda & Zwaan [8]. For fluorescence microscopy, a Leitz orthoplan microscope equipped with a high-pressure mercury lamp (HBO-200), exciter filters BG38 and UG 1, and barrier filters K430 and K460 were used with a dark field condenser. Photographs were taken on Kodak Tri-X 35 mm film.
RESULTS
Antiserum characterization W h e n tested against the i m m u n i z i n g antigen, the a n t i s e r u m d e m o n s t r a t e s three clean lines in i m m u n o e l e c t r o p h o r e s i s (fig. 1), a n d no cross r e a c t i o n to non-lens tissues can be visualized in O u c h t e r l o n y plates (fig. 2a-c). Lens antigens are u n a m b i g u o u s l y d e m o n s t r a t e d b y this s e r u m as b r i g h t fluorescence in the lens of a 96 h chick e m b r y o (fig. 5).
Lentoid analysis L e n t o i d s , identical to those illustrated b y M o s c o n a [1] f o r m with low frequency in n e u r a l retinal reaggregates. These are characterized, as are M o s c o n a ' s , b y lighter staining a n d e n l a r g e d cell nuclei, increased cytop l a s m a n d a t e n d e n c y for cells to b e c o m e p e a r s h a p e d (figs 3, 4). N e u r a l retinae f r o m 7-day old e m b r y o s were t r e a t e d in various ways, c u l t u r e d a n d analysed, utilizing imm u n o f l u o r e s c e n t techniques for lens antigens. N e i t h e r n e u r a l retina, n e u r a l retina dissociated, r e a g g r e g a t e d a n d cultured for 3 days, n o r n e u r a l r e t i n a dissociated, reaggregated, c u l t u r e d for 3 days a n d post-cultured, in the m a n n e r d e s c r i b e d b y M o s c o n a [1] gave a n y
67
i n d i c a t i o n of the presence of lens antigens (rigs 6, 7). DISCUSSION T h e structure described as " l e n s - l i k e " a n d " l e n t o i d " b y M o s c o n a , p r e s u m a b l y on the basis of its m o r p h o l o g y , does n o t in fact r e s e m b l e the a v i a n lens at a n y stage of its development. It resembles slightly a newt lens [9], b u t l o o k s m o s t like a k e r a t i n pearl [10]. These pearls are characteristically f o u n d in well differentiated e p i d e r m o i d carcinomas. The i m m u n o f l u o r e s c e n t studies r e p o r t e d here establish f u r t h e r that these p u t a t i v e lenslike structures are n o t synthesizing the u n i q u e a n d characteristic crystallins of the lens. W h a t d e v e l o p m e n t a l p a t h these structures are following is n o t clear; they are not, however, c a r r y i n g out the synthetic activities which characterize lens differentiation f r o m the fortieth h o u r on. The a p p e a r a n c e of these p s e u d o - l e n t o i d s in n e u r o - r e t i n a l reaggregate cultures treated with low c o n c e n t r a t i o n s of lens a n t i s e r u m [6], as well as the defects of the optic cup a n d b r a i n [11-15] o c c u r r i n g in e m b r y o s e x p o s e d to lens specific a n t i s e r u m continues to be p a r a d o x i c a l unless the p r e s u m p t i o n is m a d e t h a t this assay d e m o n s t r a t e s a n t i s e r u m c o m p o n e n t s t h a t are n o t visualized b y the sensitive i m m u n o l o g i c a l techniques b y which the sera were tested for cross r e a c t i o n to heterologous tissues. Technical assistants: Cindy Hambrick, Mona Tu, and Betty Ford. This investigation was supported by Atomic Energy Commission contract AT (30-1)-3825.
REFERENCES 1. 2. 3. 4. 5.
Moscona, A, Science 125 (1957) 598. - - Exptl cell res 3 (1952) 535. J anat 86 (1952) 287. - - Proc soc exptl biol med 92 (1956) 410. - - Proc natl acad sci US 43 (1957) 184. -
-
Exptl Cell Res 66
68
A. Katoh et al.
6. Braverman, M, Cohen, C & Katoh, A, J embryoI exptl morph 21 (1969) 391. 7. Sainte-Marie, G, J histochem cytochem 10 (1962) 250. 8. Ikeda, A & Zwaan, J, Invest ophthalmol 5 (1966) 402. 9. Reyer, R, J exptl zool 139 (1958) 137. 10. Alien, A, The skin, a clinical pathological treatise, p. 894. Grune & Stratton, New York (1966). 11. Burke, V J, Sullivan, N P, Petersen, H & Weed, R, J infect dis 74 (1944) 225.
Exptl Cell Res 66
12. Langman, J, Anat rec 137 (1960) 135. 13. Fowler, I & Clarke, W M, Anat rec 136 (1960) 194. 14. Langman, J & Maisel, H, Invest ophthalmol 1 (1962) 396. 15. Langman, J, Maisel, H & Squires, J, J embryol exptl rnorph 10 (1962) 178.
Received November 17, 1930