/. Embryol. exp. Morph. Vol. 50, pp. 31-45, 1979
Printed in Great Britain © Company of Biologists Limited 1979
3\
Ontogeny and localization of the
crystallins during lens development in
normal and Hy-1 (hyperplastic lens
epithelium) chick embryos
By DAVID S. McDEVITT 1 AND RUTH M. CLAYTON 2
From the Institute of Animal Genetics, University of Edinburgh
SUMMARY
A strain of chickens selected for high growth rate has been found to exhibit an anomalous
eye lens morphology indicating a failure of the normal process of growth regulation. Hyperplasia of the lens epithelium and annular pad, often with fiber formation, has recently been
described in day-old chicks of this strain, termed Hy-1 (Clayton, 1975). The earliest evidence
of this condition in this study has been found in the 11-day embryonic Hy-1 lens. Before this
time, no definitive lens abnormality could be detected histologically in the Hy-1 embryos.
However, the indirect immunofiuorescence technique had revealed early temporal and spatial
differences with regard to the lens crystallins. Antibodies, specific for 8, cathodal /?, anodal /?
and a crystallins, were applied to sections through the lens of 2^-, 3-, 3^-, 4-, 5-, 8- and 16-day
embryonic and 1-day post-hatch normal and Hy-1 chicks. 8 crystallin appears precociously
in the external layer, and cc crystallin in the prospective fiber region, of the lens rudiment of
Hy-1 embryos. Both anodal and cathodal /? crystallins are retarded, however, in their
appearance in the external layer/epithelium of Hy-1 lenses. Localization of the crystallin
classes within the lenses of the two strains continues to vary during lens differentiation until
1 day post-hatch, at which time and during late embryogenesis annular pad and epithelium
abnormalities can be frequently be seen in the Hy-1 lens. This inability to control normal lens
histogenesis thus manifests itself early as alterations in the appearance of an organ-specific
gene product, the crystallins.
INTRODUCTION
The crystallins, the characteristic structural proteins of the vertebrate eye
lens, have been found to be useful indices of the process of differentiation in this
tissue. In all vertebrates except sauropsidans, three general classes of crystallins
have been identified, each showing some heterogeneity. These are the cc, /?, and y
crystallins, the y crystallins now established as specific, when present, for the
differentiation of lens epithelial cells into lens fiber cells (Papaconstantinou,
1965; Takata, Albright & Yamada, 1965; McDevitt, Meza & Yamada, 1969;
1
Author's address (to whom all correspondence should be addressed): Department of
Animal Biology, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce
Street, Philadelphia, Pennsylvania 19104, U.S.A.
2
Author's address: Institute of Animal Genetics, University of Edinburgh, Edinburgh
EH9 3JN, U.K.
32
D. S. McDEVITT AND R. M. CLAYTON
Shubert, Trevithick & Hollenberg, 1970; McDevitt & Brahma, 1973; Brahma &
McDevitt 1974a, b). In sauropsidans, however, the y crystallin class seems to
have no physiochemical or immunological counterpart (McDevitt & Croft,
1977) and is replaced by an immunologically unique class of crystallins, the 8
crystallins (Zwann & Ikeda, 1968; Piatigorsky, 1976). The occurrence, ontogeny
and physical characteristics of the crystallins in vertebrate phylogeny have
been reviewed (Clayton, 1970, 1974).
It had been generally accepted that the vertebrate lens does not exhibit any
indications of metaplasia during its growth and development. Recently, however,
metaplasia involving a failure of growth regulation of the lens epithelium has
been observed in two genetically unrelated chicken strains selected for high
growth rate (Clayton, 1975). Histological examination of these day-old (justhatched) chick lenses frequently reveals a range of abnormalities, from doubling
of the usual single-layered cuboidal lens epithelium to the formation of short
lens fibers above the main fiber mass, associated with multiple layering in the
epithelium and annular pad. These two strains have been designated Hy-1 and
Hy-2. Preliminary observations on lenses in vivo and observation of epithelial
cells in in vitro cultures (Eguchi, Clayton & Perry, 1975; Clayton et al. \916d)
confirm that the Hy-1 and Hy-2 lens epithelial cells possess abnormal surface
properties, mitotic rate, growth characteristics and cell behavior: a de-repression
of mitosis and of contact inhibition is seen when lens epithelial cells of the two
strains in culture are compared to those of a normal, slow growing strain. In
addition, modifications of various aspects of metabolism are found, including a
differential pattern of protein synthesis and changes in mRNA stability (Clayton
et al 1976c; Truman, Clayton, Gillies & MacKenzie, 1976). Evidence suggests
that the metabolism of these cells in an epithelial hyperplastic mass is affected
by the position of the cell within the mass (Clayton et al. 1976a). In order to
determine whether any abnormalities in crystallin synthesis are intrinsic to these
genotypes or are associated only with the later stages when cells are placed in
abnormal situations, the embryonic lenses of the strain exhibiting the most
striking cytological and metabolic peculiarities (Hy-1), as well as normal
embryonic chick lenses (N), were examined using the immunofluorescence
technique. By using antibodies to specific classes of crystallins, and, where
possible, to members within a class, the ontogeny and localization of the
corresponding crystallins could be determined, and the relation of crystallin
appearance to normal and abnormal lens cell histology clarified.
MATERIALS AND METHODS
Fertilized, unincubated eggs of both normal and Hy-1 strains were either
stored at 10 °C for a maximum of 1 week until needed, or incubated for periods
varying between 2\ and 16 days at 38 °C immediately upon receipt from the
supplier. Day-old chicks of both strains as well as iced heads of freshly-killed
Crystallins in Hy-\ embryo lenses
33
normal adults were received from the suppliers, the chicks sacrificed immediately, and their lenses removed.
Preparation of antigens
a crystallin, a /? crystallin subunit of high anodal electrophoretic mobility,
a /? crystallin subunit of high cathodal electrophoretic mobility, and 8 crystallin
were isolated and prepared for use as antigens so as to obtain monospecific
antibodies, a and 8 crystallins were isolated from a total adult lens protein
supernatant (McDevitt, 1967) by repeated column chromatography on Sephadex
G-200 (Pharmacia, Uppsala), and the /? crystallins (/?l5 anodal; /?9, cathodal) by
urea-polyacrylamide gel electrophoresis as described by Truman & Clayton
(1974) and Clayton & Truman (1974). The suitability of the fractions for antibody production was determined by immunoelectrophoresis (Campbell, Clayton
& Truman, 1968) of crystallin antigens against an antibody to total adult chicken
lens proteins; a single resultant precipitin arc in the proper mobility range was
considered sufficient to proceed with immunization. Protein concentrations were
determined by the method of Warburg & Christian (1942) or by refractometry
(Campbell et al, 1968).
Preparation of antibodies
Rabbits, 2-5-3-0 kg, were either injected subcutaneously at widely separated
dorsal sites with a 1:1 or 2:1 (v/v) mixture of complete Freund's adjuvant and
total lens protein or isolated crystallins; or intravenously into the ear marginal
vein with the crystallins in 0-85 % saline, for varying periods of time. After a
positive immunologic response to the antigens was determined by titration using
Ouchterlony (1953) immunodiffusion plates, the rabbits were bled, and the
gamma globulin fraction of the serum obtained by three successive precipitations of the immune sera by dialysis against 40 % saturated ammonium sulfate
at 5 °C. This preparation, hereafter termed ' antibody', was used exclusively in
all procedures. A total of six antibodies was used in this investigation, with no
discernible cross-reactions occuring between antibodies directed against the same
class of crystallins. Antibodies against cathodal (/?9) and anodal (/?j) fractions of
/? crystallin were the same as those characterized fully by Clayton & Truman
(1974). The antibodies to a, cathodal /?, anodal /? and 8 crystallin could detect
homologous lens antigen at concentrations in the range 2-2-16 /*g/ml.
Immunofluorescence
Chick embryos of varying incubation ages (2^-16 days) of both Hy-1 and
Normal strains were dissected out, fixed in cold 95 % ethanol at 5 °C, and
processed for immunofluorescence examination as previously described (McDevitt et al. 1969). Lenses from day-old chicks were either treated as above, or
processed for immunofluorescence using a freeze-substitution method in which
material placed in absolute ethanol at — 80° is then transferred at daily intervals
34
D. S. McDEVITT AND R. M. CLAYTON
Fig. 1. Immunoelectrophoresis (Campbell et al. 1968), in 1-5% agar (Difco-Noble)
in the high-resolution buffer of Aronsson and Gronwall, pH 8-9, of total adult chicken
lens proteins against: (A) anti-total lens protein antibody; (B) anti-a crystallin
antibody; (C) anti-anodal /? crystallin antibody; (D) anti-5 crystallin antibody;
(E) anti-cathodal /? crystallin antibody. Slides stained with Coomassie brilliant blue
after formation of the precipitin arcs.
to fresh batches of absolute ethanol at - 40°,then - 20°, and finally 4°, transferred
to terpineol for 24 h, and embedded in 58° MP paraffin containing 10 % ceresin.
Embryos were staged as to days of incubation, corresponding to the stage
numbering and lens morphology of O'Rahilly & Meyer (1959).
The four types of antibodies produced, upon immunoelectrophoresis with
adult chicken total lens protein as antigen, exhibited only a single precipitin
arc, indicating their monospecificity (Figure 1A-E); this was accepted as
our criterion for antigenic specificity in immunofluorescence. The tissue section
containing eye and lens was first treated with one of the four crystallin antibodies, and then incubated, after washing, with a commercial goat or sheep
anti-rabbit gamma globulin antibody conjugated with fluorescein isothiocynate
(Miles, Inc; Burroughs-Wellcome) in the 'indirect' or 'sandwich' technique of
Weller & Coons (1954). Neither the immune antibody nor the commercial anti-
Crystallins in Hy-\ embryo lenses
35
rabbit gamma globulin-dye conjugate was absorbed with tissue powder.
Excess or unbound fluorescein isothiocyanate was removed from the antibodydye conjugates by passage through Sephadex G-25.
As a control, non-immune unconjugated rabbit gamma globlin was substituted for the unconjugated, immune rabbit gamma globulin in the middlelayer of the ' sandwich', and conjugated non-immune rabbit gamma globluin was
substituted for the fluorescein-conjugated goat anti-rabbit gamma globulin in
the top layer of the 'sandwich'. The control and experimental sections through
the eye region of the embryonic chicks and the lens of the day-old chicks were
examined with a Reichert fluorescence photomicroscope equipped with a highpressure mercury burner (HBO-200) and a tungsten lamp (12 V, 60 W). Immunofluorescence was observed and photographed under dark-field conditions.
RESULTS
{The following results are reflected in Table 1 and are based upon observations
of epithelium and fiber areas exhibiting no histological abnormality,
from at least eight lenses per age\antibody\strain\combination)
Immunofluorescence with anti-8 crystallin antibody
(A) Normal lens. At 2\ days of incubation, a positive reaction for 8 crystallin
could be found only in that part of the lens placode/vesicle closest to the prospective retina; no reaction could be found in the external layer (prospective
lens epithelium). At 3 days of incubation when the prospective fiber area is
apparent, the entire lens rudiment showed a positive immunofluorescence reaction. As lens development progressed (3|, 4, 5, and 8 days of incubation) the
fiber area, annular pad, and epithelium all remained positive for £ crystallin.
Between 8 and 16 days, however, the epithelium became negative in certain
areas, and by 1 day post-hatch, was completely negative.
(B) Hy-1 lens. At 2\ days of incubation, a positive reaction could already be
seen in both the internal and external layers of the lens placode/vesicle; this
situation persisted until the 8th day. As with the normal lens, the epithelium
became progressively less positive for 8 crystallin thereafter, until at 1 day posthatch, the epithelium exhibited no positive immunofluorescence.
It should be noted that a 'patchy' or blotchy appearance, with both the normal
and Hy-1 lenses was characteristic in the lens epithelium; this was unique to the
8 crystallin antibody in this study, and indicates that, even in advanced embryos,
not all cells of the lens epithelium are producing 8 crystallin simultaneously.
Immunofluorescence with anti-fi (cathodal) crystallin antibody
(A) Normal lens. At 2\ days of incubation, the lens placode/vesicle is positive
for cathodal /? crystallin, in both the external and internal layers, and the entire
developing lens remains positive up to and including one day post-hatch.
(B) Hy-1 lens. Only that part of the lens placode/vesicle closest to the pros-
36
D. S. McDEVITT AND R. M. CLAYTON
Table 1. Detection of crystal/ins in the embryonic chick lens
by immunofluorescence
anti-/? (Cathodal)
anti-5
Normal
Age
(days)
2*
3
Hy-1
Normal
Hy-1
c
Hy-1
Normal Hy-1
E
F
E
F
E
F
:
+
-
-
-
X
Normal
E
;
4
5
+
8
+
16
+/1 (P-H) -
F
E
F
F
+x
+
+
+
+
+
E
+
-
+
+
+
xx
E
:
+ + +
F
+
anti-a
anti-/? (Anodal)
E
;
F
+
x x ~ x x ~x
-
+
+ + + +/-
+ + +i- +
+ + +i- +
+
+
+
+ + +
+ +/- +
+ + +
E = Epithelium (includes external layer of placode/vesicle).
F = Fibers (includes internal layer of placode/vesicle).
+ = Positive immunofluorescence for designated crystallin (no distinction made for
intensity of immunofluorescence reaction or % of total area positive.
— = Negative; no immunofluorescence observed.
+ / - = positive and negative areas within same lens region (8 days and after).
pective retina {2\ days), and the prospective lens fibers (3 days) are positive
initially for cathodal /? crystallin. Not until 4 days of incubation are both the
external layer/epithelium and fiber area of the lens rudiment positive. The lens
remains positive in its entirety until 1 day post-hatch, when the fiber area
exhibits regions of positive and negative immunofluorescence.
Immunofluorescence with anti-fi {anodal) crystallin antibody
(A) Normal lens. At 2\ days of incubation, only that area of the lens placode/
vesicle closest to the prospective retina is positive for anodal /? crystallin. At 3
days, all areas of the lens rudiment become positive, and remain so up to and
including 1 day post-hatch.
(B) Hy-1 lens. Only the inner part of the lens placode/vesicle (2^ days) and
prospective lens fiber area (3 days) are initially positive for anodal /? crystallin.
Not until after 4 days of incubation do both the external and internal layers of the
lens rudiment exhibit positive immunofluorescence. This condition persists until
8 and 16 days of incubation, when positive and negative areas of lens epithelium
can be observed. At 1 day post-hatch the epithelium again appears uniformly
positive.
Immunofluorescence with anti-cc crystallin antibody
(A) Normal lens. No positive reaction to a crystallin could be observed in any
area of the developing lens of 2\- and 3-day embryonic chicks. At 1\ days, a
positive reaction could be detected in the prospective fiber area only. Not until
Crystallins in Hy-1 embryo lenses
37
8 days of incubation could a positive reaction be obtained in both epithelium and
fiber regions. This distribution remained constant in the developing lens.
(B) Hy-1 lens. In contrast to the normal lens, a positive immunofluorescence
reaction for a crystallin could be detected in the 2^-day embryonic lens, in
scattered cells in the inner part of the lens placode/vesicle. This area, as it
differentiated into the definitive fiber area, remained positive. The external layer/
lens epithelium was negative for a crystallin, however, until 5 days of incubation,
when a weak immunofluorescence reaction could be observed. At 16 days of
incubation, both positive and negative areas were evident in the lens epithelium.
At one day post-hatch, both epithelium and fiber areas appeared uniformly
positive.
Figure 2A-H illustrates the diverse immunofluorescence profile obtained when
representative normal and Hy-1 embryonic lenses were treated with the four
specific crystallin antibodies.
DISCUSSION
The ontogeny of a, 8, and to a much lesser extent, /? crystallins in the normal
chick lens has been reported by Ikeda & Zwaan (1967), Zwaan & Ikeda (1968),
Zwaan (1968), and Brahma & van Doorenmaalen (1971). In general our findings
on the normal embryonic lens are very similar to theirs; slight discrepancies
among reports from different authors are easily accounted for by small differences
in the stages recorded; by differences in the titers of the antibodies used; and in
the case of antibodies to heteropolymers, by the relative titers to different
subunit specificities of antibodies made against an entire class of crystallins.
Indeed, Zwaan & Ikeda (1968) have reported a weak positive immunofluorescence
reaction in the 54 h chick embryo lens to an antiserum directed against total
ft crystallins; this is in agreement with our results using an antibody to a cathodal /? crystallin. In this paper we report on the use of two antibodies which
distinguish between two of the /? crystallin subunits. Evidence pointing to
different times of appearance of members of a crystallin class has been previously
reviewed (Clayton, 1974), and the observations reported here on these two /?
crystallins give a direct example of their distinct behavior in immunofluorescence,
a property also reported recently by Waggoner et al. (1976). The apparent discrepancy in ontogeny of anodal /?-crystallin between their results and ours may
reflect differential antibody avidity (and therefore ontogeny), for two of the
anodal subunits.
Crystallin appearance in the development of the Hy-1 lens is strikingly
different in several aspects from the normal. The results obtained using four
different specific crystallin antibodies on both normal and Hy-1 lenses of eight
differing ages can best be summarized in tabular form (Table 1). These data
represent observations of Hy-1 lens regions with no discernable histological
abnormality. Where the characteristic hyperplasia is observed in older Hy-1
embryos (Figs. 3 and 4) the affected region assumes the immunofluorescence profile
38
D. S. M c D E V I T T AND R. M. CLAYTON
Fig. 2. Dark-field fluorescence photomicrographs of sections through the eye region
of normal and Hy-1 embryos, respectively, treated with antibody to: 8 crystallin (A
and B); cathodal /? crystallin (C and D): anodal /? crystallin (E and F); a crystallin
(G and H). At 2£ days of incubation, a positive immunofiuorescence reaction for
8 crystallin can be found in a few cells in the external layer of the Hy-1 lens rudiment
(B), while no reaction can be observed in the external layer of the normal strain (A).
A weak positive reaction for cathodal /? crystallin exists in the entire normal lens
Crystallins in Hy-l embryo lenses
at 2|- days (C), while the external layer in the Hy-l lens does not contain cathodal /?
crystallin even at 3^ days (D). The 3-day normal lens is positive for anodal /?
crystallin in all areas (E), yet the external layer of the Hy-l lens remains negative
for anodal /? crystallin until after 4 days (F). The first appearance of an immunofluorescence reaction for a crystallin can be observed at 3i days, only in the prospective fiber area (G), but a crystallin appears precociously in the prospective fiber area
of the Hy-l lens at 2\ days (H), in a few weakly-fluorescing cells (arrows). Magnification: x200, except (H): x 300. Orientation: prospective cornea to the right,
prospective retina to the left.
39
Fig. 3. Histological abnormalities frequently seen in Hy-1 late embryos: (A) 11-day embryonic lens exhibiting extrusion of the
annular pad region; (B) 16-day embryonic lens with excessive thickening of the annular pad; (C) 16-day embryonic lens with the
central and peripheral epithelium characteristically consisting of several layers folded upon one another and exhibiting varying
degrees of cellular elongation. Hematoxylin and eosin, Magnification: x 50, except (A): x 200. Orientation: cornea to the top,
retina to the bottom, ap, annular pad; ce, central epithelium; pe, peripheral epithelium.
ce
H
O
o
d
a
<
H
H
c/5
d
Crystallins in Hy-\ embryo lenses
41
of the early embryonic lens fibers. This is not surprising since the Hy-1 defect
seen by Clayton (1975) in one day post-hatch lenses expresses itself as the transformation of the epithelial cells to varying degrees of differentiated lens fibers.
The earliest observed occurrence of definitive histological abnormality in the
developing Hy-1 lens is at 11 days of incubation. Multiple layering and some
irregular extrusion of the pad area itself from the lens sphere is detectable, and
by 16 days of incubation all the gross abnormalities of the 1-day post-hatch lens
as described by Clayton (1975) may frequently be observed. This and other
commonly seen variations of the Hy-1 condition are illustrated in Fig. 3.
A-C (Hy-1 lenses, in our experience, shatter to a much greater degree than
comparable-age normal lenses when processed, accounting for the tissue debris
in the photomicrographs). Figure 4A-F exemplifies the results obtained when
Hy-1 late embryonic and post-hatch lenses, exhibiting some of the defects
described for the day-old chicken (Clayton, 1975) and seen in this study (Fig. 3),
are examined by immunofluorescence.
The mitotic index in the epithelium of the 8-day normal chick lens has been
found to decline steeply from the value observed at five days of incubation, the
rate of decline being most marked in the peripheral epithelium, which is nevertheless higher than that in the central epithelium (Persons & Modak, 1970). The
morphology of the day-old Hy-1 lens suggests that the production of excessive
epithelium is initiated after the period when mitosis normally begins to decline
significantly in embryogeny and that it fails to decline sufficiently in the Hy-1
embryos (Clayton, 1975). Evidence from incorporation studies and autoradiography using [3H]thymidine (Clayton et al. 1976 a) indicates that mitosis occurs
more frequently in the peripheral epithelium of the day-old Hy-1 chick lens
than in that of normal chick lens. The observation reported here of hypeiplasia
in the Hy-1 late embryonic lens is not inconsistent with the hypothesis that the
divergence with respect to mitotic activity between Hy-1 and normal embryos
begins soon aftei eight days, but we have no direct observation in this regard.
The peculiarities of the cells in the Hy-1 lens epithelium, as shown by the
pattern of incorporation of uridine and amino acids in autoradiogiaphy (Clayton
et al. 1976fl), strongly suggest that when a cell lies within a hyperplastic epithelial
mass, its abnormal position may act as a metabolic regulator for that cell. Thus
the activity of a Hy-1 cell is not always similar to that of adjacent cells in the
epithelial mass, but may differ sharply according to folding of the epithelium,
resulting in an epithelial cell surrounded totally (and abnormally) by other
epithelial cells. However, the striking differences in the time of appearance of
specific crystallins in the early stages described here (Table 1); the delayed
appearance of the /? crystallins in the Hy-1 epithelium; and the precocious
appearance of a crystallins in the fibers and epithelium and of 8 crystallin in the
epithelium of Hy-1 (Fig. 2.), all occur at a time when the morphology appears
to be undisturbed. This implies that some aspect of specific crystallin regulation
is subject to modification by a more constant feature of the cells than cell
42
D. S. McDEVITT AND R. M. CLAYTON
Crystaliins in Hy-\ embryo lenses
43
position. To this point, it has been observed that lens epithelial cells show a
a series of modified patterns of differential synthesis of crystallin polypeptides
in response to a wide range of signals, including cell contact, cell position and
mitotic and metabolic inhibitors, and the Hy-1 cells show genotype-specific
modifications of these responses (Clayton et ah 19766). The rate of synthesis of
the different classes of RNA, and the degree of sensitivity of protein synthesis to
actinomycin D, also distinguish normal and Hy-1 genotypes (Truman et a].
1976; Clayton et ah 1976 c). The cell membranes of 1-day post-hatch normal and
Hy-1 lenses also have been found to have strikingly different compositions when
analyzed by isoelectric focusing in the presence of 8 M urea and Triton-X
(Clayton et ah 19766). Differences in the appearances of crystallin in normal
versus Hy-1 early embryonic lenses reported herein may possibly be due to some
characteristics of the cell membranes themselves, although it cannot be ruled
out at this time that a pleiotropic phenomenon is responsible for the changes
in membrane proteins and crystallin appearance.
Fluorescent antibody procedures require a minimum concentration of an
antigen within a cell for the purposes of visualization (Coons, 1956). For this
reason it is not possible to say as yet whether it is the onset of transcription; the
stability of the specific mRNAs; and/or the rate of synthesis, turnover or assembly
of particular crystallins which are responsible for these striking differences in
the time of crystallin appearance. It would seem, however, that the regulation of
specific crystallin content of a lens cell is very strongly affected by factors
associated, causally or coincidentally, with cell membrane properties, and that
these effects on the crystallins are apparently non-coordinate. In order to
elucidate these and other problems, we are assembling evidence concerning the
rate of specific crystallin synthesis in early embryogenesis of Hy-1 and normal
genotypes (Clayton & McDevitt, in preparation). Preliminary evidence concerning differences in the pattern of crystallin synthesis discovered during
maturation of cell cultures of normal and Hy-1 lens epithelia has been
presented previously (Eguchi et ah 1975).
We are grateful to Mr J. Hunter, Mrs H. Mackenzie and Mr A. Gillies for skilful technical
assistance, and to Drs J. C. Campbell and D. E. S. Truman for helpful discussion. We are
indebted to Stirling Poultry Products, Ratho, Midlothian, and the Poultry Research Centre,
Edinburgh, for fertile eggs. This work was supported by Cancer Research Campaign (U.K.)
grant S.P. 1330/2 to R.M.C., and by a sabbatical award from the University of Pennsylvania
toD.S. McD.
Fig. 4. Dark-field fluorescence photomicrographs of sections through Hy-1 late
embryonic and 1-day post-hatch lenses, exhibiting abnormalities as in Fig. 3: (A)
11-day, treated with S crystallin antibody (ap, annular pad); (B) 16-day, 8 crystallin
antibody; (C) 16-day cathodal fl crystallin antibody; (D) 16-day, anodal /? crystallin
antibody; (E) 1-day post-hatch, treated with 8 crystallin antibody; (F) 1-day posthatch treated with a crystallin antibody. Note two areas of entrapped iris tissue
(arrows) in the hyperplastic epithelial mass of (F), also reported by Clayton (1975).
Magnification: x 300, except (A): x 200. Orientation: cornea to the right, retina to
the left.
44
D. S. McDEVITT AND R. M. CLAYTON
REFERENCES
S. K. & VAN DOORENMAALEN, W. J. (1971). Immunofluorescence studies of chick
lens FISC and a crystallin antigens during lens morphogenesis and development. Ophthal.
Res. 2, 344-377.
BRAHMA, S. K. & MCDEVITT, D. S. (1974a). Ontogeny and localization of Gamma-crystal I ins
in Rana temporaria, Ambystoma mexicanum and Pleurodeles waltlii normal lens development. Expl. Eye Res. 19, 379-387.
BRAHMA, S. K. & MCDEVITT, D. S. (19746). Ontogeny and localization of the lens crystallins
in Xenopus laevis lens regeneration. J. Embryol. exp. Morph. 32, 783-794.
CAMPBELL. J. C, CLAYTON, R. M. & TRUMAN, D. E. S. (1968). Antigens of the lens of
Xenopus laevis. Expl. Eye Res. 7, 4-10.
CLAYTON, R. M. (1970). Problems of differentiation in the vertebrate lens. In Current Topics
in Developmental Biology (ed. A. Monroy & A. A. Moscona), Vol. 5, pp. 115-180. New
York: Academic Press.
CLAYTON, R. M. (1974). Comparative aspects of lens proteins. In The Eye, Vol. 5 (ed.
H. Davson and L. T. Graham.), pp. 399-494. London: Academic Press.
CLAYTON, R. M. (1975). Failure of growth regulation of the lens epithelium in strains of fast
growing chicks. Genet. Res. (Camb.) 25, 70-83.
CLAYTON, R. M. & TRUMAN, D. E. S. (1974). The antigenic structure of chick /?-crystallin
subunits. Expl. Eye Res. 18, 495-505.
BRAHMA,
CLAYTON, R. M., EGUCHI, G., TRUMAN, D. E. S., PERRY, M. M., JACOB, J. & FLINT, O. P.
(1976a). Abnormalities in the differentiation and cellular properties of hyperplastic lens
epithelium from strains of chickens selected for high growth rate. /. Embryol. exp. Morph.
35, 1-23.
CLAYTON, R. M., ODEIGHAH, P. G., DE POMERAI, D. I., PRITCHARD, D. J., THOMSON, I. &
TRUMAN, D. E. S. (19766). Experimental modifications of the quantitive pattern of crystallin
synthesis in normal and hyperplastic lens epithelia. In Biology of the Epithelial Lens Cells
in Relation to Developement, Ageing and Cataract, vol. 60 (ed. Y. Courtois & F. Regnault)
pp. 123-136. Les Colloques de LT.N.S.E.R.M.
CLAYTON, R. M., TRUMAN, D. E. S., HUNTER, J., ODEIGAH, P. G. & DE POMERAI, D. I.
(1976c), Protein synthesis and its regulation in the lenses of normal chicks and in two
strains of chicks with hyperplasia of the lens epithelium. Docum. Ophthal., Proc. Ser. 8,27-37.
COONS, A. H. (1956). Histochemistry with labelled antibody. Int. Rev. Cytol. 5, 1-23.
EGUCHI, G., CLAYTON, R. M. & PERRY, M. M. (1975). Comparative study of growth and
differentiation of lens epithelial cells in in vitro cell culture. Devel. Growth & Diff. 17,
395-413.
IKEDA, A. & ZWAAN, J. (1967). The changing cellular localization of a-crystallin in the lens
of the chick embryo studied by immunofluorescence. Devi. Biol. 15, 348-367.
MCDEVITT, D. S. (1967). Separation and characterization of the lens proteins of the amphibian,
Rana pipiens. J. exp. Zool. 164, 21-30.
MCDEVITT, D. S., MEZA, I. & YAMADA, T. (1969). Immunofluorescence localization of the
crystallins in amphibian lens development, with special reference to the y-crystallins.
Devi. Biol. 19, 581-607.
MCDEVITT, D. S. & BRAHMA, S. K. (1973). Ontogeny and localization of the crystallins
during embryonic lens development in Xenopus laevis. J. exp. Zool. 186,127-140.
MCDEVITT, D. S. & CROFT, L. R. (1977). On the existence of y crystallin in the bird lens.
Expl Eye. Res. 25, 473-481.
O'RAHILLY, R. & MEYER, D. B. (1959). The early development of the eye in the chick.
Acta. Anat. 36, 20-58.
OUCHTERLONY, O. (1953). Antigen-antibody reactions in gels. Acta. path, microbiol. Scand. 32,
231-240.
PAPACONSTANTINOU, J. (1965). Biochemistry of bovine lens proteins. II. The y-crystallins of
adult bovine, calf and embryonic lenses. Biochim. biophys. Acta 107, 81-90.
Crystallins in Hy-\ embryo lenses
45
B. J. & MODAK, S. P. (1970). The pattern of DNA synthesis in the lens epithelium
and the annular pad during development and growth of the chick lens. Expl. Eye Res. 9,
144-151.
PIATIGORSKY, J. (1976). Subunit composition of 8 crystallin from embryonic chick lens.
/. Biol. Chem. 251, 4416-4420.
SHUBERT, E. E., TREVITHICK, J. R. & HOLLENBERG, M. J. (1970). Localization of gamma
crystallins in the developing lens of the rat. Can. J. Ophthalmol. 5, 353-365.
TAKATA, C , ALBRIGHT, J. F. & YAMADA, T. (1965). Lens fiber differentiation and y-crystallins: Immunofiuorescent study of Wolffian regeneration. Science, N.Y. 147, .1299-1301.
TRUMAN, D. E. S. & CLAYTON, R. M. (1974). The subunit structure of chick /?-crystallins.
Expl. Eye. Res. 18, 485-494.
TRUMAN, D. E. S., CLAYTON, R. M., GILLIES, A. G. & MACKENZIE, H. J. (1976). RNA
synthesis in the lenses of normal chicks and in two strains of chicks with hyperplasia of
the lens epithelium. Docum. OphthaL, Proc. Ser. 8, 17-26.
WAGGONER, P. R., LIESKA, N., ALCALA, J. & MAISEL, H. (1976). Ontogeny of chick lens
/? crystallin polypeptides by immunofiuorescence. Ophthal. Res. 8, 292-301.
WARBURG, O. & CHRISTIAN, W. (1942). Isolierung and Kristallisation des Garungsferments
Enolase. Biochem. Z. 310, 384-421.
WELLER, T. H. & COONS, A. H. (1954). Fluorescent antibody studies with agents of varicella and herpes zoster propagated in vitro. Proc. Soc. exp. Biol. Med. 86, 789-794.
ZVVAAN, J. (1968). Lens-specific antigens and cytodifferentiation in the developing lens.
/. Cell Physiol. 11, Suppl. 1, 47-72.
ZWANN, J. & IKEDA, A. (1968). Macromolecular events during differentiation of the chick
lens. Expl. Eye Res. 1, 301-311.
PERSONS,
{Received 4 August 1978, revised 15 October 1978)
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