Download The Development of the Integument: Spatial, Temporal, and

A M . ZOOLOGIST, 12:125-135 (1972).
The Development of the Integument: Spatial, Temporal, and
Phylogenetic Factors
EDWARD J. KOLLAR
Department of Oral Biology, The University of Connecticut Health Center,
Farmington, Connecticut 06032
SYNOPSIS. The development of hair, teeth, and feathers is reviewed in the context of
experimental manipulation of dermal-epidermal interactions. The inductive role of the
dermis is described, and the consequences of altering the ages and sources of the
interacting tissue components is demonstrated. The importance of examining the
developmental capabilities of these tissues after disruption of the dermal-epidermal
interface as well as in intact explants is discussed. In addition, the role of the basement
membrane in establishing and stabilizing integumental derivatives and the role of
collagen synthesis and deposition is examined.
Because of the variety of form and the
versatility of function, the skin is a favorite
research subject for developmental biologists. Consequently, the extensive work in
this area has been reviewed many times
(Billingham and Silvers, 1963, 1968, 1971;
Wessells, 1967; Sengel, 1971). In this paper, I plan to limit my discussion and to
select several aspects of skin biology of interest from the embryologist's point of
view. In addition, I would like to include a
skin derivative that is generally not included in discussions of the development
of the integument, the teeth.
Obviously, the specializations of the skin
differ in their spatial distribution in the
animal. Teeth are limited to the mandibular and maxillary regions; vibrissae, to the
snout; mammary glands, to the mammary
ridges. Furthermore, within any such general spatial distributions, finely tuned patterning often occurs. For example, in a
section taken from a mandible of a new
born mouse, the specialization of the incisor and molar tooth germs in the
tooth row include a diastema — that space
between the incisor and first molar that is
characteristic of the rodent. Similarly,
there are the familiar patterning of the
rows of vibrissae follicles on the snout
(Fig. 1) and the arrangement of haired
The original work reported here was supported
by institutional grants from the National Institutes
of Health and the American Cancer Society to The
University of Chicago.
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and glabrous regions of the skin. Obviously, ontogeny is the harmonious development of many structures into a functional
whole, not merely a list of isolated inductive events.
In addition to the apparent spatial distribution of complex structure in the embryo, it is clear that the specializations do
not appear simultaneously; there is a temporal sequence of developmental events.
Embryos age and certain structures are
related to the aging process; indeed, certain structures are diagnostic of developmental stages. Teeth and vibrissae appear
on the twelfth day of mouse gestation;
pelage follicles appear on the fourteenth
day; and so on. Epithelial and dermal tissue components in the skin of the embryo
do age, and their developmental expression is related to body locale and developmental age.
Separation of the two interacting skin
components has provided insights into the
relative contributions of the two tissues to
the initiation and maintenance of specialized structures. The importance of a continuing heterotypic tissue interaction for
the maintenance of epithelial integrity,
mitotic activity, and normal and pathological histogenetic patterns has been demonstrated repeatedly. In the absence of the
dermal component, the epithelium is not
maintained and promptly degenerates.
The dermis is less unhappy in isolation
and survives, but all too little is known
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EDWARD J. KOLLAR
FIG. 1. A section of the snout of a 15-day old
mouse embryo. Note the precise patterning of the
large vibrassae follicles. X90.
FIG. 2. A section of the snout of a 12-day old
mouse embryo. Two sites of vibrissae development
are shown. Note the condensed dermal cells and
the thickened epithelium characteristic of early
follicle development. X200.
FIG. 3. After treatment with a cold solution of
crude trypsin, the epithelium and mesodermal tissues from 13-day old snout skin are beginning to
separate. Note the dermal papillae associated with
the epithelial downgrowth. X150.
FIG. 4. A section of a 15-day old tooth germ treated
with trypsin. Note the clean separation of epithelial and mesodermal tissues. X150.
FIG. 5. The isolated epithelial and mesodermal tissues of 15-day old incisor (i) and molar (m) tooth
germs. Note also the lip furrow epithelium
(If) associated with the incisor enamel organ. X90.
FIG. 6. Follicles, keratinizing hair shafts, and sebaceous glands were formed in this experimental
combination of glabrous plantar surface epithelium
from a 14-day old embryonic footpad and the
mesoderm from the vibrissal region of a !2-day old
embryo. X750.
about possible important epithelial influences that permit or determine optimal
dermal function (Herrmann, 1960; Bernfield, 1970).
You will recognize a very common
feature of these tissue interactions (Fig.
2). The generalized epithelium responds
to a uniquely specialized dermal component, the dermal papilla. In samples of
skin taken from older embryos and subjected to a cold solution of crude trypsin (Fig.
3), the arrangement of the discrete dermal papillae to the epithelium is clearly
seen. In this preparation the epithelium is
beginning to separate. Similarly, a clearly
defined dental papilla is associated with
the tooth germ (Fig. 4).
The effectiveness of the separation is
better demonstrated by low-power photography of unsectioned material that has
been completely separated (Fig. 5). The
epithelial and mesodermal components of
the incisor and molar tooth germs are
clearly seen in the separated components
of these 15-day old embryonic tooth germs.
Enzymatic treatment at refrigerator temperatures does not dissociate the tissue into
single cells (Rawles, 1963; Kollar, 1966,
1970; Kollar and Baird, 1969, 1970a).
Rather, the basement membrane is digested and separation occurs at that level
with the result that the epithelial and
mesodermal tissues retain their threedimensional form intact.
From the experimental point of view,
the unique synthetic and structural differences between hair, teeth, and glabrous
areas of the skin such as the plantar surface skin of the foot plate provide a clearcut set of markers for any experimental
confrontation. Follicles develop in experimental combinations of glabrous epithelium isolated from the foot plate of a 14-day
old embryo and the dermal component of
the snout skin of a 12-day old embryo
(Fig. 6). The two tissues were combined
and placed in organ culture for 24 hours
to allow the tissues to establish intimate
contact. Then, the experimental tissue
combination was grafted to the intraocular
site — the anterior chambers of adult eyes.
DEVELOPMENT OF THE INTEGUMENT
FIG. 7. A well developed follicle induced from
tongue epithelium. Mesoderm from the snout of
a 12-day old embryo served as the inductive agent.
X750.
FIG. 8. Follicles induced from the tongue epithelium when snout mesoderm was present. Note the
pattern of epithelial keratinization characteristic of
the tongue. X200.
FIG. 9. A molariform tooth germ developing in an
experimental combination of lip furrow epithelium
and molar mesoderm. X250.
FIG. 10. A section of a tooth complete with dentin
and enamel matrices. Glabrous epithelium
from the surface of the foot plate of a 14-day old
embryonic mouse was induced by molar mesoderm.
X300.
FIG. 11. The dental epithelium combined with
plantar surface ilermis invades the ectopic dermal
bed in an uncontrolled fashion. X75O.
FIG. 12. A detail from Figure 11. Note the cytological changes in the basal epithelial cells. This
altered appearance is characteristic of the invading
dental epithelium combined with mesoderm from
the foot pad. X900.
FIG. 13. A keratinizing shaft can be seen in this
aberrant follicle induced by vibrissal mesoderm.
The epithelium was isolated from an incisor tooth
germ. X750.
FIG. 14. A control explant of diastema epithelium
and diastema mesoderm. XI25
127
Note that the follicle structure is complete; keratinizing hair shafts, sebaceous
glands, and a surface epithelium are all
present. When hairless epithelium from
the foot plate is challenged by a dermis
containing papillae, follicles are induced
(Kollar, 1970).
Needless to say, control explants of
plantar surface epithelium, and its homologous dermis recombined after separation
never develop hair follicles. Reciprocal exchanges of snout or dorsum epithelium
and the dermis of the foot plate develop
heavily keratinizing surface epithelium
and never specialize into hair follicles.
These data establish the inductive role of
the papilla in mammalian skin, and are
consistent with the work on avian skin indicating that the feather dermal papillae
induce feather development in normally
aptyrous chick skin (Cairns and Saunders,
1954).
Recently, I tried another experimental
confrontation in which embryonic tongue
epithelium was combined with snout dermis. These combinations were of interest
since the tongue epithelium has proved
resistant to modulation of its typically
thick keratinized epithelium into other
modes of specialization (Billingham and
Silvers, 1971). In our hands, using embryonic mouse tissue and grafting the combinations to the intraocular site, the snout
dermis is able to induce hair follicles from
the tongue epithelium (Fig. 7). However,
tongue epithelium continues to express its
characteristic pattern of keratinization;
hair follicles and the heavily keratinized
tongue epithelium appear together (Fig.
8).
Thus, it appears that when tongue or
plantar epithelium are provided with an
ectopic dermal bed, the pattern of keratinization of the surface epithelium is retained and reflects the origin of the epithelium. However, when adequate inductive
stimuli are introduced in addition, these
glabrous epithelia can express new aspects
of their genetic potential. Conserving the
specificity of surface pattern does not obviate the expression of other epithelial pat-
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EDWARD J. KOLLAR
terns.
The development of complex structure
in precise patterns is most strikingly
demonstrated by the dentition. The unambiguous incisiform and molariform shapes,
the specific differential deposition of enamel on the labial surface of the rodent
incisor but not on the lingual surface, the
unique specialization of the dental papilla
as a secretory cell population organized for
dentin secretion, and the presence of the
diastema all provide a number of challenging experimental options. Furthermore,
the anatomical similarity of the early
stages of hair follicle and the tooth germ
suggested that the initiation of tooth development be examined vis-a-vis other integumental derivatives.
The experiments were designed as before. The incisor and molar dental papillae were confronted with non-dental epithelium from the oral cavity. As the incisor enamel organ grows down into the
mandibular mesenchyme, an associated
epithelium is formed (Fig. 5). This is the
lip furrow epithelium that will split and
become the surface epithelium of the lip
sulcus. When this epithelium is isolated
and associated with incisor or molar papillae, teeth are formed (Fig. 9). The dental
structure is complete; surface epithelium,
the specialized stellate reticulum characteristic of the enamel organ, and the generalized molariform pattern have developed in
this graft (Kollar and Baird, 1970a).
These data indicated than non-dental
oral epithelium could be induced by the
dental papilla to form dental structures. In
addition, the specificity for tooth shape
apparently resides in the dental mesodenn.
Our earlier studies in organ culture (Kollar and Baird, 1969) indicated that when
recipi'ocal exchanges of dental epithelium
and mesodenn were made, the resulting
dental structure displayed the shape dictated by the source of the papilla. This
conclusion was recently confirmed in similar tissue exchanges grown as long-term
explants (Heretier, 1970) and is consistent
with similar conclusions about structural
specificity from studies of avian skin (Sen-
gel, 1971).
But, the critical test of the inductive
capabilities of the dental papilla was
whether or not dental structures could be
induced in the non-oral epithelium such as
the plantar surface epithelium. Indeed,
the epithelium from this heavily keratinized integumental site is able to function
as amelobasts, to deposit enamel, and to
maintain a characteristic dental configuration (Fig. 10). It should be mentioned
again that when the plantar epithelium
participates in tooth formation, large masses of typically stratified keratinized epithelium are produced despite participation in
new forms of specialization (Kollar and
Baird, 19706).
Control reciprocal tissue exchanges of
dental epithelium in association with other
dermal tissues were imperative since earlier workers have placed much emphasis
on the role of the enamel organ during
tooth development. In grafts composed of
enamel organ epithelium and the dermis
from the plantar surface of the foot plate,
the dental epithelium develops a stratified
keratinizing epithelium. In addition, the
ability of this epithelium to invade the
dermal bed was retained and a massive
proliferation into this ectopic dermal bed
took place (Fig. 11). The cells of this invading epithelium have a unique cytological appearance; basal cell orientation is
altered, the staining properties have
changed, and nuclear morphology is distorted (Fig. 12). In short, the epithelium
is dyskeratotic and is very suggestive of
some epithelial tumors of dental origin.
On the other hand, when enamel organ
epithelium is confronted with dermis from
the snout skin, the dental epithelium invades the snout dermis in a less random
fashion (Fig. 13). The pattern of invasion
here suggests aborted or aberrant follicle
structure. In some cases, the epithelium
under the direction of the snout dermis
organized into recognizable follicles containing hair shafts.
It appears that at the embryonic stages
examined so far, the epithelium expresses
certain stable properties with regards to
DEVELOPMENT OF THE INTEGUMENT
FIG. 15. An advanced tooth developing in a graft
of diasteraa epithelium and molar mesoderm. x 125.
FIG. 16. Another example of the harmonious development of a tooth from diastema epithelium
when confronted by molar mesoderm. X125.
FIG. 17. Three teeth developing from a pellet of
commingled cells from the diastema epithelium,
diastema mesoderm, and molar mesoderm. X125.
FIG. 18. A control organ culture of a 15-day old
incisor tooth germ grown for 8 days. X250.
FIG. 19. A BAPN-treated organ culture of a 15-day
old incisor tooth germ. This section was made
after four days of organ culture. Note the suppression of dental epithelium. X250.
FIG. 20. The recovery of a BAPNuealed culture
can be seen in this section. After four days of
BAPN treatment, this culture was moved to control medium for an additional four days. Note the
complete and harmonious recovery of the dental
structures. X250.
the kind of surface stratification it will display and whether or not it will or can
readily invade the mesoderm. Dental epithelium displays exaggerated invasiveness
129
whereas plantar and tongue epithelium do
not. On the other hand, the depth of the
invading epithelium, the definitive pattern
of invasion, and the spatial relationship
of the epithelium to the inductive papilla
appear to be determined and controlled by
the mesoderm.
Before I continue with some recent
views concerning the interaction of the invading epithelium and the modeling properties of the mesoderm, allow me to add
one further example of the importance of
the spatial distribution of inductive mesoderm.
The absence of teeth in the diastema of
the rodent mandible elicits a number of
obvious questions. Fortunately, the diastema is present and easily recognizable
from the thirteenth day of gestation. Reciprocal combinations were made with the
epithelial and mesodermal tissues from the
diastema and from molar and incisor
regions.
When dental epithelium is combined
with mesoderm from the diastema, teeth
are not formed and the dental epithelium
confines itself to a surface position and
keratinizes (Fig. 14). On the other hand,
when the epithelium from the diastema is
associated with dental mesoderm, teeth are
formed and are harmonious in all respects
(Fig. 15, 16). Apparently, the defect, if
one can properly call a functional diastema a defect, resides in the mesoderm of
that region. A suitably active inductive
dental mesoderm is excluded from that
region.
Recently, the concept of morphogenetic
field has been resurrected by Van Valen
(1970) to rationalize the order of the
tooth row. Implicit in such a theoretical
approach is the notion that gradients of
factors, both positive and negative, influence the developing structures along the
gradient distribution. Clearly within this
framework, the diastema might contain
some negative influence that prevents expression of latent inductive potential in
some cells of the diastema mesoderm.
Thus, we attempted to test the hypothesis
that negative influences in the diastema
130
EDWARD J. KOLLAR
mesoderm prevent dental morphogenesis.
In addition we wished to disrupt the
"field" by completely dissociating the dental tissues. The mesoderm from the molar
regions, the mesoderm from the diastema
region, and the diastema epithelium were
isolated and then subjected to further digestion with crystalline trypsin; the three
tissues were dissociated into a commingled
cell suspension. The suspension was centrifuged gently to prepare a pellet. After one
day in organ culture, to allow the pellet to
congeal, the pellet was explanted to the
anterior chamber. Perfectly harmonious
teeth have developed (Fig. 17) in such experimental combinations.
As a minimal conservative explanation,
it seems that the presence or absence of
teeth in the tooth row is a function of the
presence or absence of inductively active
mesodermal papilla cells. Perhaps we
should begin to analyse the tooth row, not
in terms of abstractions but in the terms of
the factors that control or direct the morphogenetic movements which bring appropriate cells into place.
The recent experiments of Stuart and
Moscona (1967), Goetinck and Sekellnick
(1970), and Ede et al. (1971) demonstrating the importance of collagen lattices in
determining the precise pattern of feather
follicles on the chick dorsum are exemplary steps in this direction. The importance of a collagen framework as a
guidance device for organizing the sites of
dermal papilla accumulation and determining the gross pattern of follicle distribution may be an exaggeration of a more general and subtle role of collagen in maintaining and controlling the spatial arrangement between the epithelium and the
mesodermal bed.
Indeed, attempts to disrupt morphogenesis with agents that disrupt collagen deposition indicate that collagen
plays a role in maintaining the spatial
relationship of the epithelial rudiment to
the surrounding mesenchyme. Grobstein
and Cohen (1965) demonstrated that the
addition of collagenase to cultures of
salivary gland rudiments prevented the
normal branching pattern of these rudiments. Similarly, Koch (1968) reported
that cytodifferentiation of the epithelial
and mesodermal cells of older tooth germs
is suppressed when explants of tooth germs
are treated with collagenase.
My laboratory has also examined the
effect of disturbing collagen deposition in
early tooth germs. We approached the
problem slightly differently; instead of collagenase, a lathyrogen, beta-aminopropionitrile (BAPN), which disturbs deposition
of the collagen fibers, was used as the disruptive agent.
When tooth germs are explanted in organ culture at the fourteenth or fifteenth
day of gestation, differentiation proceeds
and advanced stages of cytodifferentiation
are achieved by the eighth day in cultures
(Fig. 18). On the other hand, when BAPN
is added to similar cultures, the enamel
organ present at the time of explantation
regresses, and by the fourth day in vitro,
only a suggestion of a dental epithelium
remains (Fig. 19). The epithelium appears healthy and mitotic activity can be
seen often in both the epithelium and the
mesoderm. Histological preparations indicate separation at the usually tight interface between the epithelium and the dermal bed. Certainly, then, interference with
the normal deposition of collagen suppresses further differentiation, and levels of
morphogenesis already achieved are not
maintained.
This inhibition of morphogenetic expression is not permanent, however. If similar BAPN-treated cultures are moved to
control medium after four days of suppression, morphogenesis resumes, and in four
additional days in culture on control medium, the state of differentiation is equivalent to the control cultures (Fig- 20). The
recovery from BAPX inhibition is all the
more dramatic since morphogenesis is harmonious and indistinguishable from the
untreated cultures. The recovery is orderly
and apparently very rapid. These data
further emphasize the importance of collagen deposition for the maintenance of
epithelial growth and specialization. These
DEVELOPMENT OF THE INTEGUMENT
data also attest to the resiliency of developing systems.
Thus, the regional specialization of the
skin appears to be dependent on specialized dermal cell populations that direct
morphogenesis of epithelial derivatives.
How do the dermal cells acquire this property? How do they specialize in specific
terms? How do they aggregate into discrete
morphological entities? How does the epithelium contribute to dermal specialization? How do collagen lattices and stromata stabilize developmental expression?
These are challenging questions for the future.
It is perfectly clear that the morphogenesis of the skin is a series of inductive events that elicit specific histogenetic
and biochemical responses from competent
epithelia. But from analyses of the events
and processes that elicit specialization in
recombinants of skin components, it is
equally obvious that the age of the tissues
is an equally critical factor (Lawrence,
1971).
Rawles (1963) demonstrated most beautifully that the avian skin responds to inductive cues within a temporal framework.
Embryonic epithelium from the featherbearing dorsum is responsive to heterologous dermis from the scale-bearing region
for a limited time, and then the epithelium stabilizes and will produce nothing but
feathers in the presence of scale-bearing
mesoderm. The back skin is said to have
become determined; that is, it is no longer
competent to respond to a foreign inducing dermal component. Similarly, other epithelia have temporal sequences of responsiveness and stabilization. Although mammalian skin has not been examined as
thoroughly as avian skin, it seems that similar temporal patterns exist.
For example, I reported earlier (Kollar,
1966) that exchanges of epithelial and
dermal components from the vibrissaebearing snout skin and the pelage-bearing
dorsum gave two responses. Epidermis
from 11- to 13-day old snout skin responded to the presence of dermal papillae from
14-day old back skin and produced hair
131
follicles. The reciprocal combination of
dorsal epithelium and snout dermis did
not produce follicles.
Caution must be exercised, however.
Rawles warned that it is not possible to
predict results of untried combinations and
therefore any conclusion from a limited
range of experimental recombinations
must remain provisional.
Indeed, in avian skin, seemingly determined dorsal epithelium responds to inductive dermis from the beak region.
Thus, a stronger inducer may elicit new
histogenetic expression of developmental
competence from a seemingly determined
stable tissue. In addition, the response of
a given combination — especially negative
results — must be evaluated in the context
of the experimental conditions.
Sometime later I re-examined the response of the stable 14-day epithelium
from the mouse dorsum (Kollar, 1970).
New combinations were made with snout
dermis and these combinations were explanted to the nutritionally superior anterior chamber. Under these conditions, follicles were produced. Thus, determination
and competence are relative terms. Perhaps they might better be applied to investigators rather than to embryonic tissue
performance.
Similar conclusions apply to the inductive potential of the dermal component.
The quality of an inducer as a long-lived
inducer or a strong or weak inducer must
be examined in a variety of experimental
situations. For example, the dental papilla
operates as an inductive agent in experimental combinations in a wide range of
developmental stages and for long periods
of time (Main, 1966) despite passage
through monolayer culture (Kollar and
Baird, 1971). The vibrissae papillae obviously maintain the vibrissae follicle, but
they also are able to induce new follicles
when transplanted in adult rats (Oliver,
1970) in a manner which recalls the transplantation experiments of Lillie and Wang
(1944) for the mature feather. The stability of epithelial type and inductive
strength does vary from region to region,
132
EDWARD J. KOLLAR
Feather morphogenesis is suppressed by
but the stability is one of degree.
A further demonstration of the impor- mouse skin of 14 and 15 days of gestation,
tance of aging of the tissues during de- but not by skin from 13-day old embryonic
velopment is emphasized by the differen- mouse skin. The appearance of hair follitial response of commingled skin cells from cle primordia on the fourteenth day is the
various ages of chick embryos. Garber obvious morphological event associated
(Garber and Moscona, 1967) found that with the onset of the suppressive ability of
suspensions of young chick embryo skin the mouse skin.
An interesting observation was made,
cells competent to reconstruct feathers
would not do so when these young cells moreover, when the chick cells were comwere commingled with older chick embryo bined, not with whole mouse skin, but
skin cells. The older skin cells obviously with cells from either the epithelium alone
exerted some inhibitory or interfering in- or the dermis alone. Suppression of
fluence on the younger cells restricting the feather morphogenesis by mouse cells are
observed again; however, the suppressive
expression of feather morphogenesis.
The factors responsible for the inhibi- effect was noted for the 14-day old embrytion of feather morphogenesis just men- onic dermis but not for the 14-day old
tioned are unknown. But, clearly, the embryonic epithelium. The mouse epithelchanges in cell aggregability that occur ial cells do not exert a suppressive effect
with age suggest that subtle cell surface until day 15 of gestation — one day
changes may be involved. The possibility later than the dermis. Apparently, the apof some covert metabolic influence cannot pearance of the dermal papillae signals
be overlooked, of course. But, whatever marked changes in the properties of the
mediates the suppression of feather mor- dermal cell population. Thus, these studies
phogenesis, it is of a general nature since of mixtures of cells with different phylofeather development is suppressed in inter- genetic origin suggest that the age of the
species mixtures of chick and mouse skin embryo is a critical parameter to be considered.
cells.
The use of the 13-day embryonic mouse
The use of bispecific mixtures of dissociated chick and mouse skin results in cells in which the papillae had not yet
chimeric sheets of skin when reconstruc- appeared did not suppress feather mortion of the skin takes place. When compe- phogenesis and led to the startling obsertent feather-producing chick skin is com- vation that, in addition to making chimermingled with 15-day old mouse skin in ic skin, the mouse cells were able to particwhich hair follicle rudiments are present, ipate in feather morphogenesis. Chimeric
feather morphogenesis is suppressed just as feathers were constructed from chick and
in heterochronal mixtures of chick cells mouse cells. I should point out here that
(Garber and Moscona, 1964) . These data in chimeric tissues derived from chick and
suggested that the histogenesis of skin is a mouse cells it is possible to discriminate
result of many properties shared by avian the species of the cells on the basis of
and mammalian skin. In addition, the sup- staining reactions to Ehrlich's hemapressive effects of older skin on competent toxylin.
In combinations of whole chick skin and
young skin cells seems to be a common
property of older cells without regard for mouse epidermal cells, the mouse cells are
phylogenetic origin. Further analysis of the arranged in the precise pattern of the
importance of bispecific inhibition led us barb ridge (Figs. 21, 22). The size of the
to ask whether young mammalian skin cells gives the ridge different dimensions
cells would permit feather morpho- but the histogenetic pattern is harmonious
genesis, and, moreover, to examine the with the feather structure as a whole.
appearance of the inhibitory effect in the
In other areas, the combinations of
mammalian skin (Garber et al., 1968). chick skin and isolated mouse epithelial
DEVELOPMENT OF THE INTEGUMENT
FIG. 21. A section of a chimeric feather germ.
Whole chick skin cells and epithelial cells from
mouse skin were commingled. Note the darkly
staining mouse cells organized as a barb ridge-like
structure. X225.
FIG. 22. Another example of mouse epithelial cells
organized in a feather germ structure. X200.
FIG. 23. In fealherless regions of experimental
combinations of whole chick skin cells and epithelial cells from mouse skin, the darkly staining mouse
cells invade the chick dermis in a fashion reminiscent of hair pegs. X250.
FIG. 24. Mouse dermal cells accumulate at the
base of a feather germ in an experimental combination of whole chick skin cells and mouse dermal
cells. X200.
FIG. 25. An unusual interaction between chick epithelium from the mandible of a 4-day old chick
embryo and embryonic dental papilla from the
mouse. X200.
FIG. 26. In a combination similar to Figure 25, the
dental mesoderm has differentiated as odontoblasts and a thin layer of matrix has been deposited. X200.
cells from 13- and 14-day old embryonic
mice, where feathers did not develop, the
mouse cells invaded the dermis and made
epithelial structures reminiscent of hair
133
pegs (Fig. 23). There were no mouse dermal cells present, and consequently, hair
follicle development did not proceed beyond this stage. It is interesting to note
that the mouse epithelium expresses its
propensity for invasion even in this foreign
dermal bed (chick epithelium, rarely displays such a pattern) and that mouse cells
can respond to histogenetic cues from
chick dermis.
The reciprocal combinations of whole
chick skin cells commingled with mouse
dermal cells proved equally interesting.
Well-developed feathers developed in
chorioallantoic grafts of dissociated 8-day
whole chick skin and dissociated 13-day
mouse dermis (Fig. 24). Note that some of
the dermal cells from the mouse have accumulated at the base of the developing
feather.
The accumulation of mouse dermal cells
at the site of an inductive interaction in
the feather germ suggested that the mouse
cells might have participated in the induction of the feather follicle. On the other
hand, the mouse cells may have merely
homed to this site and then remained
quiescent. It was not possible to resolve
this question from data available when
these experiments were done.
More recently, however, new experiments (Coulombre and Coulombre, 1971)
have added further refinements and therefore newer insights into the problem of
bispecific tissue combinations. Using the
corneal epithelium from embryonic chicks
and mouse dermis from the flanks of 13.5
to 14.5-day old embryonic mice, feathers
were apparently induced in the absence of
competent feather-inducing chick skin
cells. In these cases, the conclusion seems
more clear:cut; the mouse cells are not
merely homing to the site of an interaction. Rather, the mouse dermal cells are
actively inducing feather germs from the
corneal epithelium.
These data once again confirm the ability of inductive events to occur across species lines. The inductive event does not
confer new information to the responding
tissue since the structure is compatible
134
EDWARD J. KOLLAR
with the genotype of the epithelium.
Feathers are induced in chick epithelium
by mouse dermal cells; hair follicles were
not formed.
My interest in the development of the
tooth prompted me to attempt combinations of oral epithelium from the chick
embryo and dental mesenchyme from the
mouse. The rationale was not entirely mad
or without justification. Dame Honor
Fell, in an early paper on the developing
mandible of the chick, described the appearance of a dental lamina in the chick
mandible. After a short time this lamina
disappears and, of course, birds are toothless. But, remember that reptiles have respectable teeth and so did the progenitor
birds.
The data I report here are very preliminary but none the less intriguing. When
the mandibular epithelium from 4- or
5-day chick embryos is combined with dental papillae from 15- or 16-day tooth
germs, a number of interesting interactions
occur. After one week of growth on the
chorioallantoic membrane of embryonated
chick eggs, the chick epithelium gives obvious evidence of interaction with the dental mesoderm (Fig. 25). Unusual topographical relationships of the chick epithelium to the mouse dermal cells occur; note
the subtle change in cytology of the basal
cells of the chick epithelium relative to the
dermal cells; note also the reaction of the
chick epithelium in the area of the interaction.
Moreover, in the best cases (Fig. 26)
there is evidence that the mouse dermal
cells, as well, are responding to the tissue
association. The mouse cells align themselves next to the chick epithelium and
differentiate as odontoblasts with the deposition of matrix material. It should be
pointed out that in all of the various combinations of dental mesenchyme with other
kinds of mouse epithelia, dentin matrix
synthesis is not initiated in the absence of
the enamel organ.
The short duration of the explanation
of these grafts would not have permitted
ameloblast differentiation. Long-term ex-
periments are now underway to examine
the possibility that avian epithelium has
the genetic information for some aspects of
tooth development but that it is not expressed. During evolution, the ability to
make teeth may have been lost — not the
genetic information; perhaps some subtle
change in the timing of developmental
events may be responsible for toothlessness
in birds.
Hopefully, new experiments will unravel this interesting aspect of bispecific
tissue interactions.
One day when the biochemists and geneticists tell us how to jump start the cell so
that any synthetic product can be made at
the whim of the investigator, there will
still be the problem of putting it all together in terms of the embryo. The building of normal embryos is a spatial and
temporal as well as a genetic problem.
Studies of the development of the skin will
undoubtedly offer many further insights
into the problems of morphogenesis. Perhaps the skin in all its bewildering diversity will be a Rosetta Stone for Developmental Biology.
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