/ . Embryo/, exp. Morph. Vol. 44, pp. 167-179, 1978
Printed in Great Britain © Company of Biologists Limited 1978
Permissive and directive interactions in lens
induction
By MARKETTA KARKINEN-JAASKELAINEN1
From the Third Department of Pathology, University of Helsinki, Finland
SUMMARY
The interactive events leading to lens formation and the developmental potentialities of
the presumptive lens ectoderm were examined in vitro. The presumptive lens ectoderm of both
mouse and chick embryos was capable of forming a lens even when isolated from the optic
vesicle before the two tissues reach the stage of close association. This lens-forming bias can
be released with favourable culture conditions and by various heterotypic mesenchymes. The
same permissive, unspecific conditions or heterotypic tissues failed to trigger lens formation
in trunk ectoderm. The directive effect of the optic vesicle was demonstrated in experiments
where it was grown in contact with the trunk ectoderm. The latter developed distinct lentoid
bodies synthesizing lens proteins. The origin of the lentoid was confirmed in interspecies
combination of chick and quail tissues. It is concluded that lens formation is governed by
a series of interactive events consisting of both directive and permissive influences.
INTRODUCTION
According to the classic concept, induction of the lens takes place during the
interaction between the retinal anlage, i.e. the optic vesicle, and the head ectoderm.
Spemann showed this interdependence in Ranafusca by removing the prospective sensory retina in the open medullary plate stage. As a consequence, lenses
failed to form and he concluded that the inductive influence of the retinal
anlage was essential for lens formation (Spemann, 1901). His discovery was
soon followed, however, by reports that similar experiments in other amphibian
species gave apparently contradictory results (Lewis, 1904; King, 1905). Most
of the experiments were reproduced, and a clear picture has emerged of the
steps leading to lens induction in amphibians (for reviews, see Spemann, 1912;
Mangold, 1931; Twitty, 1955; Grobstein, 1956; Lopashov & Stroeva, 1961;
Coulombre, 1965 and Jacobson, 1966).
(1) The prospective retina is not the only lens inductor, but merely the last
in a series of tissues that exert this inductive influence in head ectoderm during
early embryonic development. (2) Ectoderm from other sites besides the head
is capable of forming a lens. Initially, all the cells from the upper hemisphere
of the amphibian embryo possess this potentiality. With advancing development
the capacity becomes spatially restricted, the extent of the competent tissue
1
Author's address: Third Department of Pathology, University of Helsinki, Finland.
168
M. KARKINEN-JA ASKELAINEN
varying from one species to another, and also depending on the temperature
at which the embryos are reared. At lower temperatures, chemical differentiation
proceeds more rapidly than morphogenesis (Twitty, 1928). Embryos kept in the
cool before experiments will form lenses much more readily than those kept at
room temperature throughout development (Ten Cate, 1953).
Several researchers failed to obtain differentiation from the presumptive lens
ectoderm in vitro on species where it is possible even in the absence of the retinal
anlage in vivo (Perri, 1934; Woerdeman, 1941; de Vincentiis, 1949; Jacobson,
1958). It was suggested that lens differentiation depends on the inductive
influence of some other tissue besides the optic vesicle (Mangold, 1931; Liedke,
1951, 1955). Okada & Mikami (1937) substituted several tissues in the place of
the optic cup in Triturus pyrrhogaster and were able to induce a lens with the
following: nose anlage, ear vesicle, brain, heart, liver, and from younger
embryos dorsal archenteron wall, neural plate, ectoderm, mesoderm, and
entoderm of the head region. Lens formation may also be elicited by unspecific
triggers, such as salamander liver, boiled salamander heart (Holtfreter, 1934) or
alcohol-treated liver, a known inductor of anterior central nervous system
structures (Toivonen, 1949), even by treatment with acetone or alcohol, as in
Fundulus embryos (Werber, cited in Twitty, 1955). During normal development,
the first tissue to underlie the amphibian presumptive lens is the entodermal wall
of the future pharynx. While gastrulation proceeds the edge of the mesodermal
mantle, the future heart, extends to the posterior margin of the lens ectoderm.
During the neurula stage the neural folds lift the lens ectoderm from contact with
the mesoderm and the future retina evaginates from the wall of the neural tube
as the optic vesicles, which approach the ectoderm, making contact with the
presumptive lens cells. All three tissues, the pharyngeal entoderm, the heart
mesoderm and the optic vesicle, are potent lens inductors (for review, see
Jacobson, 1966). The entoderm and mesoderm gradually lose their inductive
capacity, but the retina does not. As the lens grows throughout life, it continuously requires the presence of the inductor. All 'free' lenses, which differentiate for some time even in the absence of a retina, eventually degenerate.
Although Jacobson (1956) predicts that the process of lens induction is
similar in all vertebrates, the only difference being in timing of the response of
the target ectoderm and hence in the degree of dependence upon the neural
inductor, our knowledge concerning the higher vertebrates is fragmentary.
Optic vesicle-dependent lens formation has been described in experiments
both in chick (Waddington & Cohen, 1936; Alexander, 1937; van Deth, 1940;
McKeehan, 1951; Langman, 1956) and in mouse (Muthukkaruppan, 1965).
Alexander (1937) and van Deth (1940) also reported that body ectoderm from
avian embryos 2 days old or younger responded to the inductive stimulus of the
optic vesicle by forming a lens. With advancing age of the host embryos, the
capability for lens formation was gradually lost as the cells of the posterior body
ectoderm lost their responsiveness early, while those of the head ectoderm
Interactions in lens induction
169
retained it for some time. McKeehan (1951) reported lens formation in 4-somite
embryos, not only in the head region ectoderm but also in the extra-embryonic
ectoderm.
The inductive influence of the cephalic endomesoderm of early chick embryos
was studied by Mizuno (1970, 1972). He suggested that lens induction is a twostep process, in which the hypoblast with some mesoblast cells first acts upon
the undermined epiblast until the streak stage, whereafter lens formation can
be experimentally triggered by several other tissues than the optic vesicle, such
as embryonic dorsal skin dermis, mesonephros, sclerotome, liver, and gizzard
mesenchyme, whereas retina from slightly older embryos was only a weak
inductor. Those lenses obtained in vitro were shown with immunohistological
methods to be capable of synthesizing lens-specific proteins (Mizuno & Katoh,
1972). This synthesis is detectable even before full differentiation of the lens has
taken place, which is in accordance with the development in vivo (Ikeda &
Zwaan, 1967).
In the present study, the lens forming capacity of the presumptive lens
ectoderm of mouse and chick embryos was studied in vitro. The isolated
ectoderm was grown in various culture conditions and combined with heterotopic tissues. Such permissive factors allowed lens differentiation even when the
ectoderm was isolated before making contact with the optic vesicle. The
inductive effect of the optic vesicle was studied by growing it in combination with
the trunk ectoderm of 2-day avian embryos. In the lentoids formed, the
synthesis of lens crystallins was demonstrated by immunofluorescent methods.
The possibility of cell contamination was excluded by using chick-quail
chimaeric combinations.
MATERIALS AND METHODS
Embryos
Eggs of White Leghorn chicks and Japanese quail were obtained from a local
poultry farm, and incubated in forced draft incubator at 37-5-38-5 °C. Inbred
strains A/Sn and CBA and hybrids A/CBA were used to obtain mouse embryos.
Human embryonic material was obtained from early therapeutic abortions
(Boije Hospital, Helsinki).
Preparation and culture of the tissues
All tissues were handled aseptically, rinsed and dissected in saline (mouse and
human in PBS, chick and quail in Tyrode solution) prepared with disposable
needles and grown on Millipore membrane niters (Millipore Corporation,
Bedford, Mass.). Both agarose (LTndustrie Biologique Francaise S.A., Gennevilliers, Seine) and 0-5-2-0 % agar (Difco) made up in basic salt solution proved
less favourable. The filter was placed over a hole in stainless-steel screen in
a modified Trowell-type culture (Saxen et al. 1968). When necessary, the tissue
170
M. KARKINEN-JAASKELAINEN
2
25 fj.m
100 um
Interactions in lens induction
171
fragments were coated with agar (1 %). Mouse and human embryonic tissues
were cultured in Eagle's minimal essential medium (MEM) (Gibco, New York,
cat. no. H-15), and chicken and quail embryos in BME diploid medium (Gibco,
New York, cat. no. G-13). Various other media, including McCoy's 5 a medium
(modified) (cat no. H-15), Nutrient mixture F-12 (cat. no. H-17), Medium 199
(cat. no. E-12), Waymouth's chemically defined medium (Waymouth, C :
/ . natn. Cancer Inst. 22, 1003-1017, 1959) (according to prescription: Orion
Pharmaceuticals, Helsinki), brought no obvious improvement in growth,
differentiation or survival. Although Armstrong & Elias (1968), culturing rat
eye rudiments in Waymouth's medium, observed good differentiation on adding
glutamine in concentrations of 0-1-100 mg/1., no such effect was noted in this
study, nor did addition of insulin or glucose in various concentrations improve
the results.
Addition of protein supplement to the nutrient medium proved vital for
differentiation of the lens. We used an extract of eye-free embryos of chick or
quail. In the final medium, 20 % was embryo extract and 20 % was foetal calf
serum (Microbiological Associates Inc., Bethesda, Md., U.S.A.) inactivated at
56 °C for 1 h. Plain human amniotic fluid from 6-week embryos allowed good
differentiation and so did the basic salt solutions supplemented with 40 % of
chick or quail egg yolk only. Doubling the amino acid concentration (Eagle's
essential and non-essential, Orion Pharmaceuticals, Helsinki) gave healthier
cultures, and differentiation of the avian lenses later proved to benefit when
inactivated chicken serum was substituted for foetal calf serum (Orion Pharmaceuticals, Helsinki). Streptomycin (50 jag) and Penicillin G (100 i.u./ml)
(Hoechst) were added. Cultures were grown in a humified incubator at 37 °C
in an atmosphere of 5 % CO2 in air. Cultivations seldom exceeded 6 days.
Fig. 1. The forebrain and the optic vesicles of an 14-somite chick embryo after their
separation from the overlying ectoderm and the head mesenchyme. Short treatment
with trypsin-pancreatin followed by mechanical separation.
Fig. 2. Cross-section of the forebrain region of a 22-somite mouse embryo showing
optic vesicles in close apposition to the lens placodes.
Fig. 3. Section of a well-developed lens after 5 days' cultivation of the optic rudiment
from a 22-somite mouse embryo. H & E stain.
Fig. 4. Cross-section of the forebrain region of a 16-somite mouse embryo showing
the optic vesicles separated from the presumptive lens ectoderm by a narrow interspace. The ectoderm shows no sign of placode formation.
Fig. 5. Section of an explant of the presumptive lens ectoderm cultured after
removal of the optic vesicle. The ectoderm shows a well-shaped lentoid body.
Culture time 5 days. H & E stain.
Fig. 6. Section of a lentoid body developed during 5 days' cultivation of the presumptive lens ectoderm separated from the optic vesicle before the establishment of
contact with the ectoderm. Chick embryo, 7 somites. H & E stain.
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M. KARKINEN-JAASKELAINEN
Isolation of the tissue components
Heads of the embryos were severed and kept separately from the bodies to
avoid cellular contamination. The tissues were kept at + 4 ° C for 45 min in
a solution consisting of three parts of trypsin (1:250 Difco) and one part of
pancreatin (N.F, Difco), freshly prepared. In this solution the tissues were
separated from each other by gentle manipulation and placed in serum-containing medium (Fig. 1).
Histological methods
The explants were fixed in Carnoy, embedded in paraffin, sectioned serially
at 4/tm, and stained with haematoxylin-eosin. Quail tissues were fixed in
Zenker, and Feulgen stain was applied with light green counterstain (see
Le Douarin, 1969). For immunofluorescent staining the tissues were fixed in
cold Carnoy and embedded in paraplast (Sherwood Medical Industries, Miss.).
The paraplast was removed with cold xylene, and the sections were processed
through alcohols in the cold and rinsed repeatedly at room temperature with
Ca- and Mg-free PBS. The antiserum was kindly provided by Dr Ruth Clayton
as rabbit anti-total lens antiserum lyophilizate. Normal rabbit serum in similar
dilutions was used as a control. Commercial fluorescein isothiocyanateconjugated anti-rabbit (swine) serum globulin was used (Sevac, Prague,
Czechoslovakia) with traces of amido black as counterstain (Amidoschwarz
10 B, Merck). The indirect method of Weller & Coons was employed (1954),
and the slides were incubated at 37 °C for 30 min with antiserum, rinsed and
incubated similarly with the conjugate, rinsed and mounted with glycerol-PBS.
The slides were examined with a Leitz Orthoplan microscope, and micrographs
were taken on Ilford FP-4 Panchromatic 22 din. plates. Fluorograms were
examined with a high-pressure mercury vapour lamp (Philips 200 W/4), fitted
with incident light equipment of the microscope with inbuilt dichromatic
combination of mirror (495 nm) and suppressor filter (K 510) (FluoreszenzAuflichtilluminator nach Ploem). Transmission filter 3 mm BG 12 and suppressor filter K 510 were used with additional shortwave pass-interference
filter KP 490. Exposure time was 2 min. Co-ordinates of fluorograms were
recorded and the slides rephotographed after staining with haematoxylin-eosin.
RESULTS
Establishment of contact between the ectoderm and the optic vesicle
To establish a background for the experimental studies on the various steps
of lens determination, the normal development of the interacting optic vesicle and
ectoderm was studied in the strains of mouse and chick used in our laboratory.
In a 15-somite mouse embryo the optic vesicle is approaching the ectoderm, and
a close apposition of these tissues is achieved at the 19-somite stage. At the
Interactions in lens induction
173
32-somite stage the lens vesicle is already closed and becomes detached from the
ectoderm. Similarly, close contact (at the light microscope level) between the
optic vesicle and the overlying ectoderm is established in the chick embryo at
the 10-11 somite stage, and formation of the lens vesicle is completed around
the 27-29 somite stage. Variations in rate of development were noted between
individuals and even between the eyes of the same embryo but, though frequent,
they were mostly slight and of no importance for the experiments to follow. Our
strains did not show any marked differences in the development of the optic
apparatus described earlier in mouse (Muthukkaruppan, 1965) or in chick
(McKeehan, 1951).
In vitro differentiation of the post-contact lens ectoderm
In the first set of experiments, the differentiative capacities of 'induced'
lenses were followed during culture in vitro. For this purpose, lenses with or
without their adjacent tissues were dissected from embryos that had passed the
stage of contact between the interacting tissues (above). Four types of culture
were prepared:
The optic bud with the overlying ectoderm dissected out and cultured en bloc.
The presumptive lens ectoderm separated and cultured alone.
The optic bud isolated and cultured alone.
The presumptive ectoderm combined with optic and through a Millipore
filter (transfilter culture).
With mouse tissues beyond the 19-somite stage (Fig. 2), altogether 62 cultures
were successful. Of the whole eye rudiments (optic bud plus ectoderm), 85 %
developed lenses in our conditions (Fig. 3). The corresponding figure for the
transfilter cultures was 79 % and for the cultures of isolated ectoderm 80 %.
None of the isolated optic buds showed any signs of lens differentiation.
The results with the chick tissues were basically similar (Jaaskelainen &
Saxen, 1972), except that ectoderm rarely formed a lens if isolated soon after
the stage of first contact and only acquired the capacity for independent
development around stage 19.
Experiments on presumptive lens ectoderm isolated before it established contact
with the optic vesicle
A series of experiments was next performed with donor embryos well before
the stage at which contact between the interactants is normally established
(Figs. 4 and 5). Stages close to that age were not used in the final experiments,
because isolation and separation of the components during these stages proved
difficult even after prolonged enzyme treatment, and consequently the risk of
cellular contamination increased. The four types of experiments listed in the
previous paragraph were again performed on mouse and chick tissue, and the
results are listed in Table 1.
12
EMB 44
174
M. KARKINEN-JAASKELAINEN
Table 1. Results of various types of in vitro experiments with mouse and chick
ocular tissue dissected before the optic bud had made contact with the presumptive
lens ectoderm
Lens differentiation
Type of explant (N = 30)
Mouse
Chick
Optic vesicle + ectoderm (remaining in contact)
Optic vesicle + ectoderm (in transfilter contact)
Isolated lens ectoderm
Isolated optic vesicle
25
18
7
0
26
22
0
0
Effect of heterotypic inductors and culture conditions
The demonstration that mouse lens would differentiate in ectoderm that had
been isolated well before contact with the optic vesicle was followed by various
experiments to confirm and analyse further the process. In transfilter experiments, presumptive lens ectoderm was isolated from mouse embryos at the
12- to 16-somite stage and combined with various heterotypic or heterospecific
tissues. Positive results with clearly distinguishable lenses on the ectodermal
side of the filter were obtained with the following tissues:
Head mesenchyme from 11- to 12-day mouse embryos (9/15).
Metanephric mesenchyme from 11-day mouse embryos (5/10).
Salivary mesenchyme from 12-day mouse embryos (3/10).
Pulmonary mesenchyme from 11-day mouse embryos (2/10).
Optic bud from 15-somite stage chick embryo (7/10).
Retinal tissue from 5-week human embryo (6/15).
In all these experiments, as well as in the experiments with pre-contact ectoderm, the results were greatly influenced by the culture conditions, especially
by the protein concentration of the culture medium. When cultured alone both
mouse and chick pre-contact ectoderm developed lenses in about 50 % of cases
when the sole culture medium used was human amniotic fluid. Similarly, when
foetal calf serum was replaced by chick serum, isolated pre-contact stage chick
ectoderm developed clearly distinguishable lenses in more than 50 % of the
explants (Fig. 6).
Experiments with chick trunk ectoderm as target tissues
Preliminary experiments in which the trunk ectoderm of a 4- to 14-somite
chick embryo was combined with an optic vesicle at the same stage revealed
frequently lens differentiation in the explants. To establish the origin of these
lenses and to exclude cellular contamination of either of the tissue components,
further experiments were made with reciprocal chick/quail combinations. Here,
use was made of the nuclear marker of the quail cells (see Le Douarin, 1969).
With these two species 112 successful reciprocal combinations were made between
Interactions in lens induction
175
7A
Fig. 7. (A) Sections showing the result of the experimental combination of quail
trunk ectoderm and chick optic vesicle of 8-somite embryos. (B) The cells of the
lentoid body show the quail nuclear marker. Culture period 6 days. H & E stain.
Fig. 8. Demonstration of the synthesis of lens proteins in a combination of trunk
ectoderm and optic vesicle of 8-somite chick embryos. (A) U.v. photograph of
a section of a lentoid body after treatment with fluorescein-labelled anti-total lens
serum. (B) The same section rephotographed after subsequent staining with H & E.
optic bud vesicle and trunk ectoderm. In 67 cultures the explants formed a lentoid
body or a typical lens. In the living cultures, this was visible within a day or two
as a transparent sphere. Sections usually revealed a roundish, well-delineated
body consisting of large, eosinophilic cells frequently showing elongation and
parallel orientation (Fig. 7). All the lenses formed consisted predominantly, if
not entirely, of cells with the nuclear characteristics of the donor of trunk
176
M. KARKINEN-JAASKELAINEN
ectoderm. In addition, in two cases small lentoid bodies were seen with the
nuclear structure of the donor of the optic vesicle, thus suggesting cellular
contamination or lens formation from optic vesicle cells.
Every second combination experiment had as a control on the same screen
a parallel culture of the isolated trunk ectoderm without additional tissues.
None of these showed lens differentiation. The same was true when the ectoderm
was combined with the heterotypic tissues listed in the previous paragraph or
with the following tissues: spinal cord, notochord, spinal cord plus notochord,
somites or trunk mesoderm (all from early chick embryos).
Immunofluorescence
To examine the occurrence of lens-specific antigens in lenses induced from
heterotypic ectoderm, immunofluorescent localization of lens antigens was made
as described in Methods. The antiserum stained normal embryonic chick lenses
intensely, while controls treated with normal rabbit serum remained totally
unstained. The same technique applied to lentoid bodies developed in the trunk
ectoderm revealed a distinct fluorescence strictly localized to the lens (Fig. 8).
DISCUSSION
The present results seem to fit well into the general scheme proposed by
Jacobson (1966) on the basis of his experiments on amphibian embryos.
According to this, lens induction is a multiphasic process, in which the presumptive lens ectoderm is successively exposed to several inductor tissues of
which the optic vesicle might be the last (see, however, Philpott & Coulombre,
1968). The present results show that the presumptive lens ectoderm is capable of
forming a lens even when deprived of its ultimate inductor, the optic bud,
before these interacting tissues reach the stage of close association in both avian
and mouse embryos. The observations suggest that this lens-forming bias can
be released with various, obviously 'unspecific' stimuli such as factors in chick
serum, embryo extract and by various heterotypic mesenchymes.
We cannot, however, dismiss the possibility, although slight, that the results
are due to cellular contamination. As shown in another system, kidney tubule
induction, incomplete separation of the interactants, leaving a few cells contaminating the explant, may lead to erroneous interpretations (Saxen, 1971;
Saxen & Karkinen-Jaaskelainen, 1975). In the experiments reported here, this
seems a most unlikely explanation for several reasons. In the young, pre-contact
stages especially, separation of the optic bud and ectoderm was rapid and easy,
and in the acceptable cases no contaminant cells were seen under the microscope.
Moreover, this explanation would imply that cells become detached from the
optic vesicle and attached to the ectoderm, but at this stage the optic vesicle is
already a coherent tissue and not easily damaged by enzyme treatment. Finally,
the effect of the culture condition on this 'spontaneous' lens formation would
Interactions in lens induction
111
be difficult to explain by contamination. Thus the formation of lenses out of the
presumptive ectoderm before the establishment of contact with the optic
vesicle seems to represent permissive induction (Saxen, 1977). Accordingly, the
target cells have already developed a lens-forming bias to be released by favourable, but unspecific permissive conditions. Here it should be emphasized that
the same conditions and the identical heterotypic inductors failed to trigger lens
differentiation in trunk ectoderm which evidently had not been through the
preliminary lens induction process.
The results of this last series of experiments suggest the existence of another
type of induction, recently named 'directive' (Saxen, 1977). Apparently, the
cells of the trunk ectoderm remain multipotent with many developmental
options (as also shown by previous workers dealing with the differentiation of
the skin and the appendages; see Kratochwil, 1972; Sengel & Dhouailly, 1977).
The final lens inductor, the optic vesicle, is able to direct these cells towards
lens formation and synthesis of lens-specific proteins without the early steps of
normal lens induction. This directive influence cannot be replaced by nonspecific, permissive conditions.
Our results and the scheme of multistep induction with permissive and
directive influences seem to fit well with previous observations (Introduction).
According to this view, Muthukkaruppan (1965) performed his experiments
with already conditioned or predetermined ectodermal cells and the resulting
lens differentiation was thus a consequence of permissive conditions. Experiments on very young tissues, amphibian gastrula ectoderm or chick epiblast,
on the other hand, deal with multipotent cells with several developmental
options, and need a directive induction to choose one of these, i.e. the influence
of pharyngeal entoderm, heart mesoderm or optic vesicle in the amphibian
development to form a lens and hypoblast with some mesoblast cells in the chick
embryo. Coming back to the hypothesis by Mizuno, the present results suggest
that the optic vesicle is a complete inductor, containing both first and second
step factors.
I wish to thank Dr Ruth M. Clayton, Institute of Animal Genetics, Edinburgh University,
for providing the lens antiserum. This investigation was supported by Emil Aaltonen
Foundation.
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(Received 15 August 1977, revised 24 October 1977)