A M . ZOOLOCIST, 12:13-25 (1972).
Cell Differentiation and Its Control in the Vertebrate Epidermis
B. ALLEN FLAXMAN
Temple University Health Sciences Center, Department of Dermatology,
The Skin and Cancer Hospital of Philadelphia,
Philadelphia, Pennsylvania 19140
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SYNOPSIS. Patterns of cell differentiation in the general body epidermis of vertebrates
vary from class to class and within certain classes. In fish, a multiplicity o£ non-keratinized differentiated cells are found over the body. Their presence is readily explained on
the basis o£ each arising from a distinctive precursor in the germinal layer. In
amphibians and mammals all cells form alpha keratin. In birds and some reptiles, beta
keratin is found over some parts of the body and alpha keratin is found
elsewhere. These regional differences are probably based on certain specific dermal
influences. In the epidermis of squamate reptiles, the fate of cells that have left the
germinal layer alternates between synthesis of beta or alpha keratin in a manner
unique among the vertebrates. There is evidence to suggest that feedback loops within
the epidermis coupled to specific patterns of cell behavior in the germinal layer are
important in regulating the remarkable switch in protein synthetic pathways.
A review of the ultrastructural features
of vertebrate epidermis indicates that the
morphologic changes encountered during
evolution are generally consistent with
those required for adaptation to an aqueous or land environment. In the present
discussion, I will be primarily concerned
with morphologic features of differentiation in the general epidermis that covers
the body, since this part of the epithelium
is common to all the vertebrates. The
presence of specialized epidermal appendages is variable, their homologies in dispute, and an extensive discussion would
not necessarily add to the basic purpose of
this analysis. I will also avoid incursions
into the chemistry of the products of epidermal cell differentiation. This is especially pertinent to "keratin" which, because of the peculiar insoluble properties
of the protein, has been incompletely characterized (Mercer, 1961). Instead, "keratinization" will be used only in the sense
of the sequence of morphologic changes
observed as an epidermal cell moves
This work was supported by USPHS Grant
Number 1 PO 1 CA 11536 from the National
Cancer Institute, and by USPHS Grant Number 1
PO 1 AM 15515 from the National Institute of
Arthritis and Metabolic Diseases.
toward the body surface. The specific morphologic differences between classes of vertebrates serves to point out the probable
existence of a variety of factors important
in the control of epidermal cell differentiation. After presentation of the descriptive
material, I will discuss several mechanisms
that may control differentiation, especially
in those species that display unusual complexities.
ULTRASTRUCTURAL ASPECTS OF CELL
DIFFERENTIATION
Fish
In the primative cyclostome fishes like
the lamprey, the epidermis is several cell
layers thick (Downing and Novales,
1971a,b,c). Three types of cells are present
throughout the epidermis and all are represented by specific precursors in the basal
layer. The major type is the mucous cell,
which, besides synthesizing mucous droplets, contains filaments 70 A in diameter
compacted into fibrils located peripherally
in the cytoplasm. This cell releases mucus
onto the body surface. A second type is the
club cell which is distinctive because of its
large centrally located accumulation of 70 A
filaments. Ultrastructurally, cross-sections
13
14
B. ALLEN FLAXMAN
of these filaments have suggested that they sized does not necessarily mean that a cell
are comprised of 20 A protofilaments simi- must divide for there are many examples
lar to that of alpha keratin in the higher of cells that synthesize DNA and then envertebrates. The third type is the granular ter a prolonged G2 phase (Gelfant, 1962).
cell which contains in its cytoplasm memUltrastructural studies of the epidermal
brane-bound, electron-dense granules asso- organ on the head of Morulius chrysociated with filaments about 150 A in diam- phakedion suggest that the cells show
eter. The rate of formation and fate of many features of keratinization (Sasse et
the different types of cells is not known. al., 1970). This organ is formed by an
The function of club and granular cells is infolding of the epidermis into the dermis.
likewise a mystery. Morphologic keratini- At the very surface, the cells appear to
zation does not occur in any of the three lack nuclei, and they are entirely filled
cell types. However, it has been reported with closely packed filaments. However,
that keratinization is a feature of the teeth staining for disulfide bonding is negative,
which are ectodermal derivatives (Dawson, so whether the cells are keratinized in the
1963).
same manner as mammals, for example, is
There are several ultrastructural studies still not clear (Burgess, 1956).
of the epidermis of higher fishes (Brown
and Wellings, 1970; Henrikson and Ma- Amphibians
toltsy, I968a,b; Wellings et al., 1967). In
both salt and fresh water fishes, the preSalamanders, frogs, and toads have a
dominant cells are those characterized by horny layer over the entire body (Noble,
their complement of filaments approx- 1931). By X-ray diffraction, the keratin is
imately 80 A in diameter. As these squa- of the alpha type similar to that of mammous cells move toward the surface, they malian hair (Rudall, 1947). Ultrastrucbecome natter and are ultimately exfoli- tural studies of amphibian epidermis have
ated. The number of desmosomes and fila- demonstrated that this tissue is different,
ments increases during the passage from and in a sense, somewhat simpler than that
basal layer to body surface; however, there of the fishes for only a single type of fully
is no keratinization in the morphologic differentiated cell is formed. The basal laysense. Mucous cells are also present and er seems to comprise a uniform population
arise from distinctive precursors in the of immature cells containing 70 A filabasal layer. They are fewer in number ments. After leaving the basal layer, the
than the filament-containing cells. As cells cells begin to synthesize mucus in the cismove toward the surface, the mucous drop- ternae of the smooth endoplasmic reticulets increase in size, and at the body sur- lum (Parakkal and Matoltsy, 1964). This
face they are released to the exterior. material accumulates in cytoplasmic granSquamous cells and mucous cells are con- ules, some of which are released into the
nected with one another by desmosomes. intercellular space. Another type of cytoClub cells and chloride cells have also been plasmic granule has been described but
described in the skin of fresh water fishes does not seem to be similar to the kera(Henrikson and Matoltsy, 1968c).
tohyalin of mammals (Farquhar and
It should be noted that studies with triti- Palade, 1965). At the skin surface, mucus
ated thymidine have shown that the label is no longer seen and the "horny" cells are
is incorporated into nuclei of all the epi- almost entirely filled with filaments 70-80
dermal cells above the basal layer (Hen- A in diameter. Other cytoplasmic organrikson, 1967). It was concluded that, un- elles and nuclei are absent. Whereas in
like the epidermis of higher vertebrates, fishes, mucus and filament production are
cell division must therefore occur above the function of different cells, in amphibithe basal layer. The conclusion may not be ans both functions are carried out by the
valid. The fact that new DXA is synthe- same cell. This dual synthetic ability is
4
EPIDERMAL DIFFERENTIATION AND ITS CONTROL
r
15
carried over into birds and mammals Reptiles
where it is well known that substances like
vitamin A can cause filament-producing
The members of this Class have an epicells to switch over to mucus-forming be- dermis that keratinizes fully everywhere
havior (Fell, 1957; Fell and Mellanby, over the body presumably as an adaptation
1953; New, 1965). It should be pointed to a land environment. Mucus production
out that, whereas keratinization in mam- is not found. There are, however, several
mals occurs in a uniform, apparently syn- patterns of keratinization, the most comchronized fashion, in Amphibia, neighbor- plicated being that of the squamate reping cells are found in different stages of tiles (snakes and lizards). These animals
will be discussed in some detail.
keratinization (Spearman, 1968).
Cell junctions other than desmosomes
Snakes and lizards periodically shed
have been studied in some detail in am- from the entire body surface a keratinized
phibian epidermis, and their morphology structure called an "epidermal generation"
suggests a physiological function in regulat- (Lillywhite and Maderson, 1968; Madering transport (Farquhar and Palade, son, 1965, 1966; Maderson and Licht,
1965). Cells of the outermost horny layer 1967). The epidermal generation is a comare linked by belts of plasma membrane plex structure histologically and ultrastrucfusion (zonulae occludentes) which, in turally whose genesis has been worked out
effect, bind the cells into an uninterrupted in some detail. An epidermal generation
sheet and seal off the intercellular space can be mechanically split into outer and
from the external environment. Similar oc- inner layers. With X-ray diffraction methcluding belts are found between cells of ods, the outer layer has a beta pattern
the second horny layer and in the layer similar to that of avian feather keratin
below which contains mucous granules. while the inner part has the typical alpha
The presence of the zonulae occludentes keratin pattern of mammalian hair, straprovides a morphological explanation for tum corneum, and nails (Baden et al.,
the osmotic barrier properties of frog epi- 1966; Rudall, 1947). This alterating kerdermis which impedes the movement of atinization pattern seems to be unique
water, ions, and small molecules. Similar among the vertebrates. Although only two
extensive bands of membrane fusion have types of keratin are known to be present,
not been demonstrated in the epidermis of an epidermal generation is comprised of
reptiles, birds, and mammals. The mor- several layers of morphologically different
phologic nature of barrier function in cells, all of which are derived from a morthese animals is not clear, but may be de- phologically uniform basal layer (Alexanpendent upon more massive keratinization der, 1970; Maderson et al., 1972). Immediand more layers of horny cells at the skin ately after leaving the basal layer, each cell
still shows no indication of specific differsurface.
entiation. With the passage of time and
Of interest is the fact that the horny the generation of other cells below, specific
cells of amphibian epidermis are not shed aspects of differentiation appear in the
in a continuous manner, like mammals, but different layers. The main features of the
at intervals similar to snakes and lizards fully differentiated cells are summarized in
(Dawson, 1920). This periodic sloughing Table 1. Some morphological aspects are
appears to be hormone dependent, for af- shown in Figure 1.
ter hypophysectomy, it does not occur and
The ultrastructural aspects of epidermal
horny cells pile upon one another (Adams
and Richards, 1929; Adolph and Collins, generation formation will be described for
1925; Jorgensen and Larsen, 1960, 1961). the lizard, Anolis carolinensis. The details
Shedding can be induced by injection of of generation formation are essentially
ACTH or corticosteroids. The thyroid the same for snakes with some minor differences (Alexander and Parakkal, 1969;
gland may also be involved.
16
B. ALLEN FLAXMAN
filaments, each filament about 70 A in diameter. The flattened nucleated cells lying
above the basal layer have essentially the
pat
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FIG. 1. Electron micrograph of lizard epidermis
midway through renewal phase of the shedding
cycle. Multiplicity of cell types are depicted, some
fully keratinized while others are still immature.
OEG — outer epidermal generation; IEG —inner
epidermal generation. The OEG comprises: (1)
Oberhautchen spinules (Obo) ; beta layer (Bo) ;
mesos layer (mo) ; alpha layer (ao) ; lacunar layer
(Ho) ; immature clear cells (pclo) with keratohyalin-like granules (G) . The IEG comprises: (1)
immature Oberhautchen cell (pObi) — note spinules (arrows) ; immature beta cells (pBi) — note
filament bundles (F) ; immature mesos cells (pmi) .
BC —basal cells. X6400.
Maderson et al., 1972; Roth and Jones,
1970). Shortly after shedding a complete
epidermal generation, the body is covered
with an incomplete epidermal generation
comprising a layer of basal cells attached
to the basement membrane, a single layer
of flattened, nucleated cells above, and
several layers of dead, keratinized cells.
The basal cells are typical immature cells
with an abundance of cytoplasm that contains mitochondria, free ribosomes, glycogen, etc. There are scattered bundles of
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EPIDERMAL DIFFERENTIATION AND ITS CONTROL
same ultrastructural appearance. All the
cells are connected by desmosomes. Several
layers of flat, dead, keratinized cells lying
above have been termed the alpha layer
because the characteristic X-ray diffraction
pattern is thought to originate here
(Baden et al., 1966). These cells are quite
comparable morphologically to the cornified cells of the mammalian stratum corneum (Brody, 1960; Matoltsy and Parakkal, 1968). They are anucleate, have
thickened plasma membranes, and are entirely filled with closely packed filaments of
70-100 A diameter. The next outermost
one or two layers have previously been
separated out by light microscopic criteria
as distinct from the alpha cells and are
termed mesos cells (Roth and Jones,
1967). The ultrastructural features of
these cells are not very clear, and they
have been reported as having both 20-30 A
and 70 A filaments (Alexander, 1970;
Maderson et al., 1972). At the present time
mesos cells cannot be classified precisely.
The very outermost keratinized layer is the
beta layer with spinules of the Oberhautchen on the outer surface. This layer
is an acellular unit filled with 20-30 A
filaments characteristic of beta keratin
(Filshie and Rogers, 1962; Rogers and Filshie, 1963).
Following shedding, the epidermis enters a resting phase where there is little
proliferative activity for a variable period
of time and the outer epidermal generation remains relatively unchanged. The
resting phase is followed by a renewal
phase where the outer epidermal generation is completed and part of a new generation (termed the inner epidermal generation) is also laid down. In the outer
epidermal generation, one or two layers of
lacunar cells are added. These cells are
unique in that they undergo no significant
alterations until shedding, at which time
they are immature and retain their nuclei.
The cells, however, can be induced to keratinize fully under pathological and experimental conditions where they resemble
mature alpha cells (Flaxman et al., 1968).
The generation is completed by addition
17
of a single layer of clear cells that subsequently differentiate by accumulation of
cytoplasmic granules that resemble keratohyalin ultrastructurally. In this mature,
keratinized cell the cytoplasm is amorphous with no evidence for filaments
(Maderson et al., 1972), although filaments similar to those in mature alpha
cells have been reported in snakes (Roth
and Jones, 1970).
The inner epidermal generation begins
with the emergence of the Oberhautchen,
a single layer of cells that forms spinules
on the outer surface in conjunction with
the undersurface of the clear cells above.
The Oberhautchen, along with three or
four layers of presumptive beta cells below, keratinize as the cytoplasm and spinules fill with 20-30 A filaments (Maderson
et al., 1972). Nuclei and plasma membranes degenerate. The final appearance is
that of an acellular layer filled with filaments. While cells of the beta layer are
keratinizing, immature mesos cells are
added below. Details of mesos cell formation have not been adequately studied.
Nuclei degenerate and the plasma membrane thickens, but the exact filamentous
nature of the cytoplasm remains obscure.
In snakes, membrane coating granules similar to those reported in mammalian epidermis have been described (Roth and
Jones, 1970; Matoltsy and Parakkal, 1965) .
The next cells added form several layers
of alpha cells. These cells keratinize in a
manner similar to that of mammalian stratum corneum as the cytoplasm fills with
filaments ranging from 70-100 A in diameter. Shedding of the complete, mature outer epidermal generation occurs shortly after this stage leaving behind an incomplete
new outer epidermal generation.
It should be stressed that all cells, immediately after leaving the germinal layer,
appear the same, and only 50-70 A filaments are found in the cytoplasm. Specific
differentiation begins only at a later time
in the renewal phase. At all times, germinal cells appear the same and give no clue
as to the type of daughter cell to which
they will give rise. The latter can be pre-
18
B. ALLEN FLAXMAN
dieted, however, from histologic identification of the shedding cycle stages.
The problem of control of cell differentiation in the epidermis of the squamate
reptiles will be discussed in detail below. It
should be pointed out that hormones
have been found only to influence the time
relationship between the resting and
renewal phases of the shedding cycle, but
not to have any direct bearing on specific
aspects of cell differentiation within each
part of the cycle (Maderson et al., 1970).
In other reptiles, both alpha and beta
keratin are present in the epidermis, but
are located in different regions of the body
surface rather than in the manner of the
squamate reptiles. In turtles, for example,
all cells of the carapace may have beta
type keratin and resemble lizard beta cells
ultrastructurally, whereas cells of the epidermis of the neck resemble alpha cells
(Baden and Maderson, 1970; Alexander,
1970). Likewise, in crocodile scales, while
all kcratinization of the outer scale surface
is of the beta variety, the inner scale surface is of the alpha type (Baden and Maderson, 1970). Thus, unlike the squamate
reptiles, where both types of keratinization
are found everywhere over the body surface, the keratinization pattern in turtles
and crocodiles varies according to a particular region, different regions showing different patterns.
Birch
Keratinization of the avian epidermis in
general resembles that of the mammalian
epidermis with certain differences. The
basal cells contain only 60-80 A filaments
and produce daughter cells whose pattern
of differentiation is uniform (Matoltsy,
1969). As the cells move toward the body
surface the filaments aggregate at the cell
periphery. Centrally, lipid droplets and socalled multigranular bodies appear. At
higher levels, keratohyalin granules accumulate at the cell periphery. During
keratinization, most cell organelles degenerate leaving only filaments, keratohyalin,
lipid, and thickened plasma membranes.
The fully keratinized cells of the lower
layers of stratum corneum comprise a central lipid mass surrounded by a fibrous
cortex. The outermost keratinized cells are
very thin and have no central lipid mass.
The synthesis of morphologically discrete
lipid masses makes keratinization in the
avian epidermis different from that of
mammals. In most other aspects, however,
the systems are similar. It should be mentioned that the keratin pattern of the surface epidermis of birds is of the alpha
variety. Beta keratin is found in feathers.
As for crocodile scales, beta keratin is
present on the outer surface of leg scales,
while alpha keratin is found on the inner
scale surface and hinge region (Baden and
Maderson, 1970). Thus, although both
types of keratin are represented in the
birds, they appear in different parts of the
integument and not in an alternating vertical pattern characteristic of the squamate
reptiles.
Mammals
All keratinized structures derived from
the mammalian epidermis have the alpha
X-ray diffraction pattern (Baden and
Maderson, 1970). In the general body epidermis the process of keratinization is similar to that of birds and only a single type
of keratinized cell results (Brody, ]9r>9a,b,
1960; Matoltsy and Parakkal, 1968). The
germinal cells contain 50-70 A filaments
that seem to increase in number as differentiation proceeds. However, large lipid
droplets do not appear. During keratinization, nuclei, mitochondria, ribosomes, and
other organelles degenerate. Keratohyalin
granules appear among the filaments, the
plasma membrane thickens, desmosomes
become altered, and membrane-coating
granules discharge their contents into the
extra-cellular space. The fully keratinized
horny cell is a bag consisting of a tough
cell wall enclosing abundant filaments in
an amorphous matrix. The stratum corneum plays an obviously important role as
a barrier which acts to keep essential elements within the body (especially water)
EPIDERMAL DIFFERENTIATION AND ITS CONTROL
while keeping out a large variety of substances found in the environment.
THE CONTROL OF EPIDERMAL CELL
DIFFERENTIATION
From the foregoing comparative descriptive material, it is apparent that the epidermis of some vertebrates is more complicated than that of others. Whereas in
mammals, birds, and amphibians only a
single type of mature cell is formed over a
specific body region, in fish and squamate
reptiles a multiplicity of cell types are
found. Although the cell variety in fish can
be explained on the basis of specific precursors in the basal layer, in squamate reptiles no such variety of precursors has been
demonstrated. It would seem that the control of epidermal cell differentiation may
be more complex in some vertebrates than
in others. In this section I will discuss controls of differentiation under the following
general categories: (1) epithelial-mesenchymal interactions; (2) cell-substratum relationships; (3) intercellular communication; and (4) mitosis.
Epithelial-Mesenchymal Interactions
Several excellent reviews have covered
this subject (Billingham and Silvers, 1963;
Wessells, 1967). At the outset it should be
noted that although such interactions
have been rather conclusively shown to be
important during embryogenesis and in
the adult state, the mechanism remains
completely unknown. Mesenchymal influences act over a distance of 50-100^ but no
chemical mediator has ever been isolated
(Grobstein, 1961, 1967). If epithelium and
mesenchyme are separated by a Millipore
filter, collagen and mucopolysaccharide accumulate under the epithelium, and it has
been proposed that an interaction with
these macromolecules results in the observed epithelial behavior patterns (Grobstein, 1968; Kallman and Grobstein, 1965,
1966). There is also growing evidence to
indicate that in early development mesenchyme only facilitates certain pre-
19
programmed patterns of epithelial cell
differentiation (Bischoff and Holtzer, 1969;
Wessells, 1964ft, 1968). It is possible,
therefore, that certain pathways of differentiation have already been determined
before specific morphologic aspects become
evident. Most epithelial-mesenchymal work
has been concerned with influences on epidermal organogenesis such as feather, hair,
and scale development of which the keratinization process is only a part. In the formation of appendages, specific differences
in keratinization appear, but these are
superimposed on a genetic background
where the capacity to keratinize is already
an established epidermal cell attribute.
It is clear that the early embryonic ectoderm can follow pathways of differentiation leading not only to epidermis but also
to neural tissue. Experiments have shown
that development into epidermis is the
more primitive state that appears earlier in
development (Holtfreter, 1968). The actual histogenesis of the epidermis may not
be dependent on mesenchyme (see below
under cell-substratum relationships), for
isolated amphibian ectoderm will form epidermis at the gastrula stage when mesoderm has not yet appeared (Holtfreter,
1968). It is not clear, however, whether
mesenchymal influences initially determine
the range of specific patterns of ectodermal cell differentiation. For example, in
animals whose epidermis keratinizes, no
studies have been carried out to determine
precisely when the capacity of cells to keratinize appears in development. When asyet morphologically undifferentiated chick
ectoderm is separated from its underlying
mesenchyme and grown in vitro, many
cells die but some keratinize (McLoughlin, 1961a, 1963; Sengel, 1956). Thus, the
capacity for keratinization must already
have been present although the cells
showed no detectable evidence of it.
Once the basic pattern of cell differentiation has been established, it appears that
mesodermally derived tissue can effectively
modulate cell expression. For example,
embryonic chick ectoderm combined with
gizzard mesenchyme in vitro will produce
20
B. ALLEN FLAXMAN
mucus instead of keratin (McLoughlin,
1961&, 1963). Such an effect may be mediated by extracellular mesenchymal material (Marin and Sigot, 1965). The modulation of keratinization is reversible and
seems to depend on continuous mesenchymal stimulation (McLoughlin, 1968). This
is consistent with work using vitamin A as
a modulator of epidermal differentiation
where mucus formation ceases and keratinization reappears when the vitamin is removed from the environment (Fell, 1957;
Fell and Mellanby, 1953; and FittonJackson and Fell, 1963).
Keratinization in adult vertebrates, although fully established as an epidermal
cell attribute, continues to be influenced
by underlying dermis. Regional differences
in keratinization are probably accounted
for on this basis. For example, the full
keratinization process (cornification) of
body epidermis ceases abruptly at the border of the mucosa of the human lip which
is characterized by incomplete keratinization. Nevertheless, the mucosal cells come
to resemble epidermis if transplanted to a
dermal site on the body surface (Van Scott
and Reinertson, 1961). Dermis has been
shown to influence the thickness of epidermis and stratum corneum in guinea pigs.
Thin ear epidermis transplanted to sole of
foot dermis develops a thick stratum corneum characteristic of sole (Billingham
and Silvers, 1967). Departures from keratinization, such as are normally seen in
human sebaceous glands, most likely are
the result of dermal influences, for the cells
that make lipid are fully capable of keratinizing in response to injury (Strauss and
Kligman, 1958). Further evidence that
dermis can suppress keratinization comes
from studies in vitro where previously nonkeratinizing human epidermal basal cell
tumors fully keratinize in the absence of
surrounding connective tissue (Flaxman
and Van Scott, 1968). It appears, therefore, that from early embryonic development throughout adult life, keratinization
is the dominant form of epidermal cell
expression, but it can be modified or suppressed by factors that originate in the der-
mis. Although not yet studied, it seems
likely that the regional differences in the
type of keratin found in bird scales and in
the epidermis of non-squamate reptiles
also have their basis of expression in specific dermal differences.
Although it might be expected that cell
differentiation in the complex lizard epidermis might be under a major dermal
influence, experimental studies in vitro
have shown that this is not the case
(Flaxman et al., 1968). Isolated resting
phase epidermis grown on Millipore filter
continues to show highly organized, synchronized behavior in which all the visual
cell types are produced. Other evidence in
support of this epidermal independence
comes from studies of skin wounding in
lizards. Normally, wounding is followed by
local disappearance of generation formation and proliferating cells give rise only
to daughter cells which form alpha keratin. During regeneration, the local epidermis re-enters the shedding cycle in conjunction with the rest of the body epidermis before the dermal component of the
scales has been reconstituted (Maderson,
personal communication). Studies in
mammals have also indicated that some
epithelial tissues are more independent of
dermis than others with respect to expression of differentiation (Billingham and
Silvers, 1967).
Epidermal Cell-Sub stratum Relationships
It has generally been observed that epithelial cells must attach to a substratum in
order to survive. The epidermal cell in
vivo rests on a basement lamella whose
origin has been thought to be from epidermis itself, from dermis, and from both tissues (Berliner, 1969; Hay, 1964; Kallman
and Grobstein, 1965, 1966). A certain
amount of circumstantial evidence has suggested that basement lamella is at least
partly made of collagen (Hay, 1964). Since
the mechanism of epithelial-mesenchymal
interactions has not been discovered in
the form of a diffusible chemical mediator,
it has been suggested that the interaction
EPIDERMAL DIFFERENTIATION AND ITS CONTROL
between epithelium and the collagen macromolecule might be involved (Grobstein, 1968). Certainly, organogenesis can
be severely disturbed in tissue culture in
the presence of the enzyme collagenase
(Grobstein and Cohen, 1965). Perhaps
regional differences in chemical composition and physical properties of basement
lamella have some role in regulating epidermal cell differentiation.
In early studies where isolated embryonic chick epidermis was grown in vitro, the
epithelium did not survive and show histogenesis unless recombined with its mesenchyme (Dodson, 1963, 1967; McLoughlin,
1961a; Wessells, 1962). Subsequent studies
revealed that survival and development
would occur in the absence of mesenchyme
if the basal cells achieved attachment to a
substratum like Millipore filter or collagen gel (Dodson, 1967; Wessells, 1964a).
Studies with adult human epidermis in
vitro have confirmed the work with chick
epidermis (Flaxman et al., 1967). Attachment to a substratum seems to be a necessary prerequisite for cell proliferation and
appears additionally to inhibit the keratinization process since detached germinal
cells keratinize. How the substratum inhibits keratinization is unknown; it appears
that epidermal cells can only be influenced
while attached to the substratum. For example, work with vitamin A indicates that
this substance modulates differentiation
only by acting on cells before they leave
the basal layer (Wessells, 1967). The importance of events in the germinal layer as
relates to subsequent cell differentiation
will be discussed below.
Cell Communication
Electrical communication mediated by
direct cell-to-cell passage of small molecules has now been demonstrated for a wide
variety of cells. The excitement centering
around this discovery concerns obvious implications for the control of cell behavior
and differentiation. Although not proved
conclusively, communication is thought to
be mediated via special cellular junctions
21
known as "nexuses" or "gap junctions"
whose ultrastructural features have been
described by numerous authors (Brightman and Reese, 1969; Goodenough and
Revel, 1970; Revel and Karnovsky, 1967).
Communication is absent or diminished
between some, but not all types of malignant cells, and a deficiency of nexal connections has been reported (Lowenstein
and Kanno, 1967; McNutt and Weinstein,
1969; Sheridan, 1970). Among the vertebrate epidermal systems, that of amphibians has been studied, and it has been
demonstrated that the cells do communicate
(Lowenstein and Penn, 1967). Following
a wound, communication is disrupted and
reappears shortly after the healing epithelial edges meet. The role or roles that
communication might play in the regulation of differentiation in the simpler systems like those of birds and mammals is
not clear. Examination of the more complex lizard epidermis, however, affords some
observations that strongly indicate a role
for communication in the regulation of
patterns of keratinization.
Lizard skin can be grown in organ culture where the epidermis continues to
form complete, morphologically normal
epidermal generations in which the beta
and alpha keratins are formed in an alternating pattern (Flaxman et al., 1968).
Following treatment with trypsin the outer
layers can be removed leaving behind a
single layer of basal cells on the dermis.
These cells will readily proliferate. If the
outer layers are removed at a time when
the basal cells were forming daughter cells
that would keratinize as alpha cells, only
alpha cells subsequently differentiate and
the beta pattern does not reappear. Likewise, there is evidence that following trypsin treatment when beta cells were being
generated, such cells continue to be formed
and the alpha pattern fails to reappear. It
seems likely that there is some sort of
feedback between the outer and inner epidermal layers that plays a role in the
switch back and forth from alpha to beta
keratin. The feedback probably works on
22
B. ALLEN FLAXMAN
cells in the germinal layer as will be dis- begin differentiation rather than leaving
cussed in the next section. That the feed- immediately on completion of mitosis
back represents a form of cell communica- (Greulich, 1964). Although such an event
tion is suggested by some unpublished may be thought of statistically in terms of
morphological data. While ultrastructural maintaining the steady state, it may also be
studies have failed to demonstrate any possible that during the time a cell renexal junctions during the relatively quies- mains in the basal layer, certain important
cent resting phase of the shedding cycle, decisions are made regarding its fate. For
nexuses appear in large numbers during example, will it divide again or will it
the renewal phase and have been found differentiate and if so, how will it differenlinking not only cells of the same type, but tiate? For epidermal systems with a unialso cells of different types (Flaxman, un- form pattern of keratinization, there may
published data). Since the renewal phase be no a priori reason to think that the
represents a time where important changes message to keratinize in a particular manin cell differentiation take place, it seems ner would be dependent on the length of
reasonable that these changes could be reg- stay in the basal layer. When analyzing the
ulated, at least in part, via intercellular squamate epidermis, however, with its inherent complexities, such a possibility may
communication.
not be far-fetched. Moreover, in the epithelium of the duodenum, where several
Mitosis
cell types are found on the villi, studies
The relationship between cell differenti- already have indicated that there may be a
ation and mitosis has been a subject of significant relationship between the length
past and continuing debate. While there of sojourn in the proliferative zone
are examples of heightened cell differenti- of the crypts and the subsequent path of
ation appearing after DNA synthesis is differentiation of cells on moving out of
blocked (Silagi, 1969), there are certainly the crypts (Thrasher, 1970). Since an ininstances where an extremely rapid rate of traepidermal feedback has been demonproliferation is perfectly compatible with strated in lizard epidermis, perhaps the
full differentiation such as during hair feedback acts at the level of the basal layer
growth. In the present section I am not so to regulate the fate of cells that go on to
much concerned with rates of proliferation differentiate. Some recent unpublished obas I am with the fate of cells immediately servations of the behavior of cells following mitosis in the basal layer of the lizard
following a mitosis.
epidermis may have some bearing on this
In studies that have been concerned matter.
with defining the mechanism of the steady
state in renewing tissues, most attention
During the renewal phase of the lizard
has been paid to the relationship between epidermis, the outer epidermal generation
cell loss at the epithelial surface and how is completed by addition of the lacunar
cells leave the germinal layer. Following and clear layers. The inner epidermal genmitosis, there are several possible fates for eration is begun and the Oberhautchen,
the resulting cells. Both can remain behind beta, mesos, and alpha layers are formed.
to divide again, both may leave and diff- If animals are injected with a single pulse
erentiate, or one may remain behind and of tritiated thymidine just prior to emerone may leave (Greulich, 1964; Leblond et gence of the lacunar cells and the skin
al., 1964). Probably all three situations biopsied every 12 hours until the end of
exist in maintaining the steady state. It has renewal phase, no cells with labeled nuclei
been demonstrated in esophagus, for ex- emerge from the basal layer for 3-4 days
ample, that following mitosis, many cells (Flaxman and Maderson, unpublished
may remain in the germinal layer for a data). During this time the labeling index
prolonged period of time before leaving to in the basal layer increases to 40-50^ of all
EPIDERMAL DIFFERENTIATION AND ITS CONTROL
cells, representing a 4-5 fold increase over
that present at the time of injection of
tri dated thymidine. The first labeled cells
above the basal layer appear coincident
with the emergence of the first mesos cells.
With the formation of the mesos and alpha
layers, both of whose cells have labeled
nuclei, the labeling index gradually falls.
The observations can only be interpreted
as showing that cells destined to differentiate as mesos and alpha cells arise from a
group of cells that have been generated in
the basal layer and remain there, perhaps
dividing again, for up to 4 days before
emerging. It seems reasonable to assume
that this striking delay in the emergence of
labeled cells may well represent a time
when the future pathway of differentiation
of cells is being influenced. Although it is
not clear whether mesos cells are merely
very flat alpha cells or whether they have
the filamentous composition of both alpha
and beta cells, their release coincides with
the time when keratinization switches
from the beta to alpha pattern. It has been
postulated that the emergence of new expressions of cell differentiation in embryonic systems coincides with the occurrence
of specific or so-called "quantal" mitoses
(Bischoff and Holtzer, 1969; Holtzer et al.,
1968). Although not exactly comparable,
perhaps the changing pattern of keratinization in lizard epidermis has a similar relationship to cell division. The switch-over
from beta to alpha keratin may depend on
cells dividing a critical number of times
in the basal layer before they emerge.
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The Following Abstract was Printed Incorrectly in Volume 11 #4, November
1971.
LEE EHRMAN, State University of New York at
Purchase and The Rockefeller University
Frequency-dependent mating success (in Drosophila
and elsewhere?).
The mating success of the carriers of a genotype
is sometimes dependent upon the frequency o£ this
type relative to other competing genotypes; this is
especially true of a repetitively mating sex, for
instance, the mating advantage accorded rare Drosophila males. Sexual isolation, the weakness or lack
of mutual attraction between opposite sexes from
different populations, takes precedence over frequency-dependent mating advantages and where
sexual isolation is strong, these advantages do not
exist. Sexual isolation is manifested between species
while frequency-dependent reproductive successes
are intrapopulational. When the latter are present
in Drosophila, their bases may be olfactory. Experiments are underway testing for such frequencydependency in nature since all previous work has
been performed in the laboratory. (Supported by
U.S.P.H.S. Career Award 2K03 HD09033-07.)
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