Developmental Morphology of the Skin and Hair Follicles in Normal and in 'Ragged' Mice
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Developmental Morphology of the Skin and Hair
Follicles in Normal and in 'Ragged' Mice
by j . SLEE 1
From the A.R.C. Animal Breeding Research Organization, Edinburgh
WITH TWO PLATES
R A G G E D (Ra) is a semi-dominant mutant gene which was first reported by
Carter & Phillips (1954). The adult morphology, the genetics, and the em-
bryology of the mutant mice were described by Slee (1957 a, b). It was found that
adult ragged heterozygotes (i?a+) had sparser coats than normal, many of their
hair follicles being incompletely developed and non-functional. Ragged homo-
zygotes (RaRa) were almost naked. Most of their pelage hair follicles were either
absent, or abnormal and non-functional. Ra-\- embryos could be identified from
16 days' gestation by the retardation of their sinus hair-growth. RaRa embryos
were characterized from 13 days' gestation by the occurrence of a generalized
subcutaneous oedema which persisted until birth, and also by retardation in the
development of their sinus hairs and follicles. Pelage follicle primordia appeared
at the normal time (14 days' gestation) in Ra-\- and RaRa embryos but sub-
sequently developed slowly in RaRa embryos, especially when the oedema was
pronounced.
This paper describes the development of the skin and the pelage follicles in
normal and in ragged mice, from the 14th day of gestation until the 21st day
after birth. These data are linked with the previously described anomalies of
adult ragged mice in an attempt to establish the causes of the anomalies and
their times of first appearance. Moreover, it was hoped to test the concept of
Falconer, Fraser, & King (1951), which supposed that each of the hair-fibre
types in the adult mouse was associated with a specific time group of developing
follicles in the embryo or neonatal mouse. On this theory, the deficiency of
zigzag hairs found in adult Ra-\- mice might be associated with early postnatal
follicle defects, whereas the slight excess of guard hairs and awls should stem
from a higher than normal follicle density in the late embryo. The near-nakedness
of adult RaRa mice could derive from anomalies at many stages of follicle
development.
From the observations of Griineberg (1943), Falconer et ah (1951), and Gibbs
(1941) it was thought that the initiation of hair follicle primordia would begin
1
Author's address: A.R.C. Animal Breeding Research Organization, Field Laboratory, Dryden
Mains, Roslin, Midlothian, Scotland.
|J. Embryol. exp. Morph. Vol. 10, Part 4, pp. 507-29, December 1962]508 J. SLEE — D E V E L O P M E N T OF SKIN AND
in the 14-day embryo and continue until 6 or 8 days after birth. The growth of
early initiated follicles would proceed simultaneously with the initiation of later
ones, but, after initiation was completed, follicle development was expected to
continue until the completion of the first hair-cycle at about 3 weeks of age.
The rate and time of follicle initiation and development were compared in
normal and ragged mice. The differentiation and growth of skin layers (epider-
mis, dermis, and adiposus) were also studied, being relevant to some anomalies
found in adult RaRa mice, viz. epidermal hyperplasia and absence of the adipose
layer.
Furthermore, Slee (1957a) found that in normal mice neighbouring follicles
were usually in the same stage of the hair-cycle at any time, whereas adjacent
hair follicles in Ra-\- mice tended to be out of phase with each other. Con-
sequently the patterns of hair-growth were ill-defined. Information on the cause
and development of this anomaly was expected to assist in an understanding of
the normal hair-cycle. Since some form of hair-growth cycle occurs in many,
perhaps in nearly all, mammals, the problem of its control is worth investigation.
MATERIAL AND METHODS
The data described in this paper come from observations on 34 embryos and
103 suckling mice of ages from 13 days' gestation until 21 days' post-partum.
The mice were drawn from a small, but not deliberately inbred, population.
At each age -\--\-/Ra-\- comparisons were made between littermates. RaRa
mice were in some cases compared with Ra+ littermates, but + + littermates
were rarely available since throughout most of the experiment viable RaRa
mice were not obtainable from intercross matings and had to be produced by
backcross matings of the type Ra+% x RaRad (Slee, 19576). The mortality of
RaRa mice, especially those showing high-grade expression of the gene, was
higher than normal both before and after birth. Therefore the RaRa mice used
tended to be of the 'low-grade' type which showed little subcutaneous oedema.
Two to five mice of each genotype ( + + , Ra+, and RaRa) were killed at each
of the following ages: 17- and 18-day embryos, and 0, 1, 3, 4, 5, 7, (9), 12, (15),
18, (21) days' post-partum. At the parenthesized ages no RaRa mice were avail-
able. Two skin samples (from the neck and sacrum) were taken from the dorsal
surface of each mouse and used for the preparation of whole mounts or sagittal
sections. For the whole mounts the skin was shaved, scraped free of connective
tissue, fixed in Bouin's fluid, and stained in Delafield's haematoxylin. For the
sagittal sections the skin was shaved, fixed in Bouin's fluid, sectioned at 7 n, and
stained with Delafield's haematoxylin and eosin. In addition, 10 whole embryos
aged from 13 to 16 days' gestation were sectioned transversely at 10 /i and
stained as above.
The skin preparations were used to obtain the following measurements:
follicle density, hair-fibre density, follicle length, rate of follicle development,
and thickness of epidermis, dermis, and adiposus. Follicle density was measuredH A I R FOLLICLES IN MICE 509
on whole mounts of skin up to 4-6 days after birth, after which the density and
size of hairs and follicles were too great for accurate counts to be made. There-
fore, follicle density was also measured independently on the sagittal skin sec-
tions throughout the whole age range, by counting the number of follicles per
standard microscope field. This method included small follicle primordia which
might not be scored on the skin whole mounts. Follicle development was
assessed by the appearance of such features as sebaceous glands, hair canals,
hair shafts, &c. Follicle lengths and the mean thickness of the skin layers were
measured directly on the sagittal sections.
Measurements of the stratum corneum layer of the epidermis were discarded
because varying amounts of this layer were lost during the preparation of the
skin. Subsequent references to 'epidermis' are therefore not inclusive of the
stratum corneum. The dermis layer of the skin was defined from the underside
of the epidermis to the adipose layer. The adiposus extended from its junction
with the dermis to the panniculus carnosus muscle layer.
RESULTS
1. Skin morphology
Epidermis
The data here amplify the descriptions of normal mouse epidermis develop-
ment due to Gibbs (1941) and Hanson (1947). Comparable information on
the development of the epidermis in
ragged mice is juxtaposed.
The changes in thickness of the
40
35
ft, Neck
epidermis which occur during hair- ho
follicle development and during the Sc 25
progress of the first hair-cycle are | 20
~ 15
V
shown in Text-fig. 1. In general there ' - • " " ~ * * - »
10
was no significant difference between 17 01 3 5 7 9 11 13 15 17 19 21
Pre -natal Post-natal Aqe in days
ragged and normal mice before birth
and up to 4 days afterwards. How- Sacrum
ever, after 4 days RaRa epidermis
and to some extent Ra+ epidermis
both decreased in thickness signifi-
cantly less than normal.
The cytological diiferentiation of
normal mouse epidermis varies con-
siderably. It develops from the early 17 01 11 13 15 17 19 21
Pre-natal Post-natal Aqe in days
embryonic periderm to form about
4 rows of Malpighian cells in the TEXT-FIG. 1. Mean epidermal thickness (ex-
d udtag
15-day embryo. Then it thickens and L
becomes more proliferative, and
the other typical cell layers appear before birth. Moving proximally510 J. SLEE —DEVELOPMENT OF SKIN AND from the stratum corneum the following layers can be distinguished: stratum lucidum, stratum granulosum (containing keratohyalin granules), and the Malpighian layer and stratum intermedium which comprise together 2-5 rows of columnar cells. As the epidermis decreases in thickness some time after birth, it also dedifferentiates. During this process the stratum granulosum and stratum intermedium eventually disappear and the number of cell rows in the Malpighian layer is reduced to one or two. The morphology of the Malpighian cells also alters in that their nuclei become smaller, more irregularly shaped, and pyknotic during dedifferentiation, and their orientation becomes roughly pavement-like rather than columnar. Maximum dedifferentiation is normally established by 15-18 days after birth when the epidermis consists only of stratum corneum and 1-2 rows of Malpighian cells. This is typically the state of adult epidermis during the resting stage of the hair-cycle. Summarizing then, as the epidermis reaches maximum thickness in the late embryo and neonatal mouse, differentiation of the separate layers is most complete: as its thickness diminishes dedifferentia- tion occurs, during which the stratum granulosum and stratum intermedium virtually disappear. In ragged mice the cycle of differentiation and dedifferentia- tion is similar, but the process of dedifferentiation takes place later and more slowly than normal and never becomes so complete, especially in ragged homozygotes. Even as late as 18-21 days after birth stratum intermedium cells and keratohyalin granules are usually visible in ragged mice. A striking abnormality which occurs in a few RaRa mice is that of localized hyperplasia of the epidermis. Usually seen during the proliferative phase of the epidermal cycle, i.e. in late embryos or 0-4-day-old suckling mice, these regions of hyperplasia are caused by gross thickening of the stratum granulosum and the Malpighian layer, with an increase in the number of cell rows. Compared with an average epidermal thickness of 30-35 /x, the hyperplastic regions may be 140 fi thick in extreme cases. In contrast, some RaRa embryos showed abnor- mally thin and cytologically undifferentiated epidermis. But in these cases the thin epidermis was associated with (and possibly caused by) a high-grade of subcutaneous oedema. In the affected zones the dermis/adipose region was dis- rupted and occupied by oedematous fluid. The morphology of Ra-\- epidermis was generally similar to normal. Dennis The dermis does not become histologically distinguishable from the adipose layer until about 1 day after birth. From this age until 21 days, dermis thickness, in normal and ragged mice, fluctuates from 65 to 125 /x. These fluctuations seemed random and there were no consistent differences between the three genotypes + + , Ra+, and RaRa. Adiposus Throughout most of the period of these observations there were very large
HAIR FOLLICLES IN MICE 511
and highly significant differences between + + , Ra+, and RaRa mice in the
thickness of the adipose layer (Text-fig. 2). Although the + + adiposus achieved
a far greater thickness than in Ra+ mice, the latter maintained its thickness
for longer so that the two coincided
in the later stages. RaRa adipose 450
Neck
thickness was nil or very small 400
throughout the period of the obser- 3,350
vations. Z 30°
| 250
I 2 00
2. The initiation and density of
150
hair follicles 100
Early embryos 50
Estimates of follicle density on 17 3 5 7 9 11 13 15 17 19 21
the body were made during the Pre-notol Post-natal Aqe in days
early stages of follicle development TEXT-FIG. 2. Mean thickness of the adipose
by counts of follicle primordia on the layer. The bar lines denote ± one standard
error, where these were large enough to appear
perimeters of successive transverse on this scale. Adiposus thickness in the sacral
sections of whole embryos. Three region varied in a fashion closely similar to that
oedematous embryos (putatively of the neck region, in all genotypes.
RaRa, see Slee, 19576) and three non-
oedematous embryos (putatively Ra+) between the ages of 14 and 16 days were
compared in this way. In each case follicle density was significantly lower in the
RaRa embryos (means: 7-61,9-16, and 12-00follicles per section) than in their non-
oedematous littermates (corresponding means: 9-66,16-00, and 15-63). Although
no allowance was made for the mechanical stretching effect of subcutaneous
oedema upon the skin, these results did suggest that early pelage follicle initiation
proceeded slowly in high-grade RaRa embryos. However, there was no evidence
that follicle initiation began any later than normal. In Ra-\- and RaRa embryos,
as in normal embryos, pelage follicles first became visible at 14 days' gestation.
Late embryos and suckling mice
Follicle density changes were estimated from whole mounts of skin, up to
4 days' post-partum, on the type of material shown in Plate 2, figs. L-N.
Independent data obtained from sagittal sections over the whole period of
development are shown in Text-fig. 3. Both sets of data indicated that differences
in follicle density between + + and Ra-\- mice were small and often of doubtful
significance. Although + + embryos showed a higher follicle density than Ra-\-
embryos at 17 days' gestation, at some stages after birth Ra+ mice seemed to
achieve a density as high as or higher than normal. This was clearer in the data
from skin whole mounts, where for the first 2 or 3 days after birth Ra+ follicle
density was significantly greater than normal. RaRa mice showed generally a
lower follicle density than + + or Ra-\- mice, but at a few stages the differences
were not statistically significant. After birth follicle density was always much512 J. SLEE —DEVELOPMENT OF SKIN AND
greater on the sacrum than in the neck region for all three genotypes. This is in
agreement with earlier data on adult follicle density in these mice.
Fluctuations in follicle density will result from the interaction of two oppos-
ing processes: (i) the initiation of new follicles, tending to increase density,
(ii) growth of the animal with consequent expansion of the skin, causing de-
creased follicle density. The steep rise in density near birth must indicate a peak
rate of follicle initiation at this time, especially in the sacral region. The decline
in density from about 5 days after
birth will be due to body growth,
but it also indicates that follicle ini-
tiation has slowed down or ceased
after this age. Since + + and Ra-{-
mice grow at similar rates between
birth and weaning (Slee, 19576) direct
comparison between them is valid.
It therefore appears that their rates
and times of follicle initiation are
fairly similar, although Ra-\- mice
may be rather slower before birth
and slightly faster immediately after-
wards. RaRa mice, however, grow
more slowly than normal and so
their rate of follicle initiation must
be more subnormal than is apparent
7 9 11 13 15 17 19 21
Pre-notol Post-natal Aqe in days
from the graphs of follicle density.
Both direct observations and the
TEXT-FIG. 3. Mean follicle density estimated
from sagittal sections of skin. The bar lines density trends in Text-fig. 3 suggest
denote ± one standard error, wherever these that the initiation of new follicle
were large enough to appear on this scale. primordia had ceased in all genotypes
soon after 5 days' post-partum. This
is rather earlier than was reported by Gibbs (1941).
The tendency for Ra-\- follicles to become denser than normal for a transient
period soon after birth may be related to their subnormal size. Each of these
follicles occupies a smaller surface area of skin than the larger normal follicles,
and so a higher density may be attainable for physical reasons. When later a
large number of new primordia are initiated in normal mice, the situation seems
to become equalized or reversed (Text-fig. 3).
Follicle group formation
Different phases of follicle initiation in the mouse are associated with typical
patterns at each stage of development. This phenomenon, which occurs in most
mammals, has been described in mice by Gibbs (1941). She pointed out that
follicle groups were transient in the mouse. Groups of 3 (trios) appeared by theHAIR FOLLICLES IN MICE 513
4th day after birth; then as more follicles were initiated the groups became
obliterated. In the present work it was thought that the changes in follicle density
and rates of initiation might be associated with changes in the group patterns.
Group formation was, therefore, studied on the skin whole mounts used for
follicle density estimates (Plate 2, figs. L-N).
Stage Age
0
o o o 14-17 day
o
embryo
o oo
0 n
17 day embryo-
oOo birth
oQo
ooOoo o oOoo
Birth-3 days'
ooOO000 ooO°° post-partum
ooQoo o ooOOo°
o o O o o oooOoo ooOOooo 4
0O000O00 oooOOO° 0 0 onwards
TEXT-FIG. 4. A diagrammatic representation of the normal
stages of follicle group formation and subsequent oblitera-
tion during development of the follicle population.
From these observations the normal process of follicle group formation can
be summarized (Text-fig. 4). There was an initial period when the early initiated
follicles were uniformly spaced, a period near birth when clearly defined groups
were visible, and a period during which the initiation of many new follicles in
between existing groups caused the formation of rows of follicles in which
separate groups were not distinguishable. Groups seemed to be formed rather
earlier in these mice than was reported by Gibbs. Ragged mice showed some
differences. In i t a + mice, especially immediately after birth when follicle
density tended to be high, there was less-clear grouping of follicles. The groups
in RaRa mice were always less clear than normal, but those groups that were
distinguishable often persisted much longer. This seems to confirm that the
rate of follicle initiation was subnormal.
5584.4 L1514 J. SLEE —DEVELOPMENT OF SKIN AND
3. Hair-follicle morphology
The development of the normal hair follicle has been well described by, for
example, Stohr (1903) and Pinkus (1958). The sequence of development in the
young mouse is very similar to that of the adult hair-cycle (Dry, 1926; Chase,
Rauch, & Smith, 1951). In essence it comprises the following stages: (i) the
formation of a primordium from the basal layer of the epidermis, (ii) down-
growth with development of the
follicle shaft and bulb and in-
vagination of the dermal papilla,
(iii) differentiation of an inner
sheath and hair cone, (iv) formation
of the hair fibre and the accessory
sebaceous gland, (v) hair prolifera-
tion, (vi) shortening of the follicle
accompanied by distal or upward
movement of the completed hair
fibre, (vii) follicle quiescence. This
series of events is equivalent to the
first hair-cycle and it spans the
period from the 14-day embryo,
when follicle initiation first com-
mences, to the 3-weeks-old wean-
ling, when all follicles have become
quiescent.
The development of ragged and
0 1 3 5 7 9 11 13 15 17 19 21
normal follicles is compared below.
Pre-natal Post-natal Aqe in days The abnormal growth retardation
TEXT-FIG. 5. Mean follicle length estimated on of Ra-\- follicles and the almost
sagittal sections of skin. The bar lines denote totally arrested development of
± one standard error wherever these were RaRa follicles are shown in terms
large enough to appear on this scale.
of mean follicle length in Text-
fig. 5. All of the postnatal between-genotype differences shown were highly
significant except between + + and Ra-\- after 15 days. In + + mice peak
follicle length (the period of maximum hair proliferation) was reached at 9 days
in the neck region and at 12 days on the sacrum. Ra-\- follicles, growing more
slowly than normal, attained a lesser mean maximum length, and were later
than normal in reaching their peak (viz. 13 days sacrum, 15 days neck). It may
be significant that normal follicles reached their maximum length earlier at the
neck than at the sacrum, whereas in Ra-\- mice this sequence was reversed. If we
assume that the retardation in Ra-\- follicle growth was greater at the neck than
at the sacrum, this evidence falls into line with data on the adult ragged mouse,
where hair-follicle abnormalities were typically most severe in anterior regions.HAIR FOLLICLES IN MICE 515
During the period of retraction in follicle length, after 12 days, the delayed
progress of Ra-\- follicles again becomes apparent. They commence to shorten
slightly later than normal follicles and so their mean lengths coincide with nor-
mal during this period. At the time of maximum follicle length (9-13 days)
a few of the largest Ra+ follicles were longer than any normal follicles although
the mean length of all Ra+ follicles was subnormal. These large follicles had
apparently been initiated early and were at no time retarded in development. It
seems likely that they were guard-hair-producing follicles; because such follicles
are normally initiated first (Falconer, Fraser, & King, 1951), and because in
adult Ra+ mice the guard-hair fibres tend to be longer than normal (Slee,
1957a). At most periods of development Ra-\- follicles were clearly shorter than
normal; but their length distribution was more dispersed, since there were
many more small follicles than normal and a few more very large follicles. Apart
from their decreased size and growth rate, other important abnormalities of
ragged hair follicles were observed. These are best indicated by a day-to-day
comparison with the normal.
Initiation of the earliest Ra-\- and RaRa follicles commences at the normal
time—in the 14-day embryo.
Between 14 and 17 days' gestation there is little difference from normal except
in those RaRa embryos which show high-grade subcutaneous oedema (Slee,
19576). Here there is some retardation of early follicle development from the
young primordial stage.
By 17-18 days the normal embryo has some follicles with bulb, papilla, and
inner-sheath development. There are fewer such in Ra-{- and none in RaRa
embryos. In normal and Ra-{- embryos the follicles of the neck region are
already more advanced morphologically than those at the sacrum. This dif-
ference persists normally, but it is confounded somewhat in Ra-\- mice and
markedly in RaRa mice with the tendency for all follicle anomalies—including
growth retardation—to be more severe in anterior regions.
After birth many new follicles are initiated. In Ra+ and especially in + +
mice, follicle development becomes out of phase in that the new follicles are
behind the early initiated follicles in their size and stage of development. In
RaRa mice the new and the older follicles are all still in primordial stages, and
follicle development soon begins to fall markedly behind normal (Plate 1). In
some individual RaRa mice many of the follicle primordia tend to be abnormal
in structure. The epithelial cells forming the outer margins of these follicles are
not grouped in the even radial arrangement found in normal mice, but they are
scattered in the region of the primordial cell mass imparting to it a rather diffuse
and indefinite structure (Plate 2, figs. 0, P). No dermal papilla anlagen are
visible at the bases of these RaRa follicle primordia. Often the mesoderm is dis-
rupted by the presence of subcutaneous oedema, and possibly this prevents
the formation of the papilla which in turn could affect the organization of the
follicle primordium.516 J. SLEE —DEVELOPMENT OF SKIN AND
Between 1 and 3 days after birth + + follicles develop fast, hair fibres are
formed in some follicles, and eventually no primordia remain. In Ra+ mice
development of the largest (earliest initiated) follicles proceeds normally, but
the smaller follicles which were initiated postnatally lag behind comparable
normal follicles (Plate 1). Ra-{- follicles are, therefore, more out of phase than
normal in their stages of development. In RaRa mice the follicle population is
still limited to primordia of varying sizes.
Between 5 and 7 days after birth + + follicles become closely in phase in their
stage of development, as the later initiated follicles grow sufficiently fast to catch
up those initiated early. All normal follicles are fully functional hair-producers
by this stage. Late initiated Ra-\- follicles are, by comparison, grossly retarded,
whereas the early follicles are normally developed. The Ra+ follicle population
therefore becomes out of phase in its development (Plate 1). Many large follicles
become curved or bent in shape, and may be orientated at abnormally acute
angles in relation to the skin surface (Plate 1, fig. E). These distortions seem to
be associated particularly with regions where the skin thickness is abnormally
small (see Text-fig. 2).
From 7 to 12 days + + follicles remain uniform in length, orientation, shape,
and phase of growth. Hair proliferation and follicle size are maximal. The
Ra~\- follicle population presents a wide variety of types from normally developed
hair-producing follicles to others so retarded as to consist merely of strings or
clumps of epithelial cells. It is clear that the follicles which are grossly retarded
at this stage will never become functional, since the phase of follicle-growth is
almost over (Text-fig. 5). These incomplete follicles, none of which possesses
differentiated inner sheaths, hairs, or sebaceous glands, obviously represent
the non-functional follicles which are found in adult Ra+ mice (Slee, 1957a).
In RaRa mice there are still many follicle primordia remaining at this stage.
There are also incomplete follicles similar to those in Ra+ mice. The largest
of these follicles possess differentiated shafts and bulbs but no inner sheaths,
hairs, or sebaceous glands. They are still much smaller than normal.
Between 12 and 15 days hair-growth ceases and the follicles begin to retract
upwards. This process commences rather earlier in normal follicles than in even
the functional follicles of Ra-{- mice. The accompanying decrease in skin thick-
ness also tends to occur slightly later in Ra-\- mice. The Ra+ follicles of dis-
torted shape become even more grossly curved and bent during this stage of
development (Plate 2,fig.J). Those follicles which were well grown, but later than
normal in commencing to shorten, appear to be compressed as the skin thickness
diminishes. They maybe out of phase with the skin contraction even though this
is slightly delayed. In RaRa mice the largest of the follicle-like structures also
become curved or bent in some degree (Plate 2, fig. K). The most advanced of
these structures have by this stage developed hair canals which are frequently
plugged with keratin. No hair-producing follicles were seen.
Between 18 and 21 days the normal follicles completely retract and enter theHAIR FOLLICLES IN MICE 517
resting stage closely in phase with each other. The largest Ra+ follicles follow
suit; those Ra+ follicles which are slightly retarded but functional behave
similarly, but enter this stage slightly later than normal. Many of the incom-
pletely formed follicles also undergo length retraction (but to a lesser extent than
normal) during this stage. The largest of the incomplete follicle structures in
RaRa mice show a tendency to retract slightly but very much later than normal.
The numerous incomplete follicles show little change in morphology. The RaRa
follicle population is now so grossly abnormal as to be difficult to describe in
terms of the normal stages of development.
Hair formation Setaceous gland formation
Birth A«. Ir. An», ^ 18 Birth An. In ,jOyS ,. 18
— A* NSocrum
eck
1-RaRa
J
TEXT-FIG. 6. A diagram showing trends in follicle development
as they differ between normal and ragged genotypes. The points
are approximate mean values based on counts obtained from
sagittal sections of skin.
Text-fig. 6 shows in quantitative terms the degree of retardation in structural
development suffered by ragged follicles. The two characteristics used—hair-
fibre and sebaceous gland formation—are both closely related to the stage of
development of the follicle as a whole. In general these data confirm the
descriptive information given above.
The whole range of morphological abnormalities in the hair follicles of young
ragged mice can now be assessed and summarized as follows:
(i) Follicle initiation commences at the normal time in Ra-\- and RaRa
mice. Some early initiated Ra-\- follicles are normal in their rate and
final extent of development.
(ii) The next initiated Ra+ follicles are still functional, but their growth is
retarded to varying degrees so that they are out of phase with each other
in development and in completion of each stage of hair-fibre formation.
(iii) The slow growth-rate of Ra+ follicles is associated with a subnormal
mean maximum size and a delay in the time of reaching maximum size.518 J. SLEE — DEVELOPMENT OF SKIN AND
(iv) The shape and orientation of many Ra-\- follicles and the largest RaRa
follicles are abnormal,
(v) Later initiated Ra-\- follicles and nearly all RaRa follicles are grossly
retarded in development, and they remain as incomplete non-functional
aggregates of epithelial cells,
(vi) In general the retardation of RaRa follicles is more severe than that of
Ra+ follicles, but most of the consequent abnormalities are qualitatively
similar. One additional RaRa anomaly is apparent failure of dermal
papilla formation in some early follicle primordia.
DISCUSSION
This discussion is based on the present observations and also on data from
two previous papers on ragged mice (Slee, 1957 a, b). Much of the earlier material
can only be discussed now in the context of the juvenile abnormalities.
Juvenile follicle abnormalities and the adult syndrome
The basic abnormality of the pelage in ragged mice is a general restriction of
hair-follicle development and hair-growth in young suckling mice. In Ra-\-
mice this restriction is most severe in late initiated follicles (postnatal) and it
does not affect the early initiated follicles. The result is that some follicles re-
main incompletely developed, and the rest of the population becomes out of
phase in its development. Growth restriction is more extreme in anterior than in
posterior regions and everywhere its intensity is much greater in RaRa mice
than in Ra+ mice.
These abnormalities link directly with those of adult ragged mice, viz. in
Ra+ mice: incomplete development of many follicles, virtual absence of zigzag
hairs in anterior regions of the body and subnormal density of zigzags in posterior
regions, structural hair defects, the existence of transitional hair types, and out-
of-phase progress of adjacent follicles through the hair-cycles; in RaRa mice:
incomplete development or absence of nearly all follicles, causing virtual hair-
lessness.
According to a hypothesis of Falconer, Fraser, & King (1951) hair follicles
initiated in the normal 14-17-day embryo later produce guard hairs, those
initiated between 17 and 19 days produce awls and auchenes, and those initia-
ted after birth produce zigzags. Since it is the late initiated follicles whose
growth is most curtailed in juvenile Ra-{- mice, one would expect, on the hypo-
thesis of Falconer et al., that the zigzag hairs would be affected most in the
adult mice. This was, in fact, the case. Moreover, the existence of transitional
fibre types in the adult is consistent with the time sequence of follicle develop-
ment being upset in the juvenile mouse. On the same theory, the fact that adult
Ra+ guard hairs tended to be longer than normal and the awls and zigzags
shorter than normal again fits with the juvenile data, since a few of the early
initiated Ra-{- follicles were larger than normal at some stages, whereas all theHAIR FOLLICLES IN MICE 519
other functional follicles were smaller than normal. Despite its general sufficiency,
the hypothesis of Falconer et al. does seem at variance with the present data in
one respect. It was found in adult Ra-\- mice that the density of guard hairs and
awls tended to be greater than normal. However, the follicle density of Ra+
mice is never greater than normal before birth, when guard hair and awl
follicles would supposedly be initiated, but may slightly exceed normal im-
mediately after birth when zigzag follicles would ex hypothesi be initiated. One
must conclude that the high density of guard hairs and awls in adult Ra+ mice
does stem from the supranormal postnatal follicle density. Probably the
Falconer-Fraser-King hypothesis is correct for the normal mouse but in Ra-\-
mice the time sequence of follicle development, although in essence the same,
may have been slightly disorganized by changes in the relative growth rates of
different follicle types. Consequently, discrete periods of follicle initiation are
not precisely associated with production of the same fibre types as normal.
Nevertheless, it is almost certain that guard hairs, awls, and zigzags are, in
Ra+ mice, still related to the same order of follicle initiation: early, moderate,
and late as in normal mice. It is probably true of nearly all mammals that the
earliest formed follicles become largest and produce the largest types of fibre.
Such differences in follicle size, associated with their time of initiation, may
actually determine the type of fibre which each type of follicle shall produce.
We can now conclude, with Falconer et al., that certain discrete periods of
follicle initiation are each associated with the production of a certain fibre type
in the normal mouse. In the Ra-\- mouse the order of formation of different
follicle/fibre types is unaltered, but the times allotted for each period of follicle
initiation are assumed to have been slightly disorganized. If the sequence of
follicle initiation and the production of a heterogeneous fibre population are
interrelated processes it is not surprising that their disruption in i?a+ mice
causes changes in fibre-type proportions, defects of hair structure, and the
production of transitional hair types. All these anomalies occur consistently in
adult Ra-\- mice.
The abnormalities of the RaRa mouse are so extreme that the relationship
between follicle and fibre-type anomalies cannot be meaningfully discussed.
Partial follicle agenesis or severe follicle-growth retardation in the juvenile
mouse is paralleled in the adult by virtual nakedness, except for the existence of
a few fibres unclassifiable into the normal morphological types.
Causes of the anomalies in hair-follicle development
The pelage defects of ragged mice were generally more severe in some body
regions than in others. For example, Ra-\- juvenile follicle development was
more retarded in the neck region than on the sacrum. As a probable consequence
Ra+ adult mice lacked almost all zigzag hairs on the head, neck, and anterior
regions of the body; whereas on the posterior dorsum the frequency of zigzags
was almost normal. Between anterior and posterior areas there seemed to be an520 J. SLEE —DEVELOPMENT OF SKIN AND evenly graded increase in density of zigzag hairs. In RaRa mice the situation was similar in that rather more of the few hair fibres produced existed in posterior regions of the body. It may be significant that the least abnormal areas are those which, according to Dry (1926), cease hair-growth last during production of the first pelage in the normal mouse. For example, in the head and neck regions maximum follicle size is normally reached by 9 days after birth (present data) and hair-growth ceases 17 days after birth (Dry); whereas on the sacrum maxi- mum follicle size is not reached until 12 days and hair-growth ceases at 19 days. Present data suggest that follicle initiation commences at similar times on the neck and sacrum, but that follicle development proceeds more slowly and finishes later on the sacrum. It seems, therefore, that the effects of the Ra gene are least severe where there is most time available for follicle- and hair-growth. Moreover, the Ra gene acts least strongly against the earliest initiated follicles (the guard-hair- and awl-producers) which again have the most time available for development. These facts suggest that a primary effect of the Ra gene is to retard rather than prevent the development of many hair follicles. This effect might then interact with the time factor so that follicles with least time for development—which need to grow fastest—would be most severely affected. Nevertheless, follicle-growth retardation may be so extreme as virtually to prevent all development (as in many RaRa follicles). In such cases the effects are indistinguishable from those of growth prevention. If we assume that retardation of follicle-growth is a major effect of the Ra gene, the next step is to ask how this effect is mediated. Two possible processes could be postulated: (i) mechanical interference with follicle-growth, (ii) slowing down of the rate of division of the follicle-forming cells. Presumably any interference of the first type would be visible histologically. Then the only possible suppressor of follicle development would be the pre- sence of subcutaneous oedema. In high-grade RaRa embryos between 15 and 17 days' gestation, subcutaneous oedema can be widely distributed (Slee, 1957&). Skin depths of up to 600 fx between the epidermis and the panniculus muscle layer may be entirely permeated by oedematous fluid, causing complete dis- ruption of the mesodermal layer. Despite the apparent isolation of the epidermis, follicles are initiated normally in these areas. This may be possible because the initiation of follicle primordia is caused by migration of the epidermal Mal- pighian cells, and not at first by an increased rate of mitosis (Balinsky, 1950). However, one would expect there to be clear effects on follicle development and, in fact, follicles developing over sites of oedema are usually atypically small even for RaRa embryos. Many such follicles do not possess dermal papillae, perhaps as a result of the permeation of the dermis by oedema. The develop- ment of the follicle blood-supply is also likely to be impaired. But there are serious objections to regarding oedema as the main cause of growth retarda- tion in ragged follicles. First, oedema has not been seen (macroscopically or microscopically) in any postnatal Ra+ mice, and it appears very rarely and
HAIR FOLLICLES IN MICE 521
inconspicuously in Ra-\- embryos. Nevertheless, consistent coat defects, stemming
from defects of postnatally formed follicles, are typical of all Ra-{- adult mice.
Secondly, oedema in RaRa embryos is of variable extent in different individuals
and is hardly visible in certain anatomical regions, e.g. the head. Despite this,
similar skin and follicle anomalies occur in all body regions in all adult RaRa
mice. Thirdly, if oedema acted as a physical agent to interfere with follicle-
growth one would expect that the few follicles which did mature would occur in
isolated groups at points of local failure of the mechanical interference. In fact,
the functional follicles occur singly and they are evenly spaced from each other.
It is concluded that subcutaneous oedema does not play a primary role in re-
tarding the development of ragged mouse follicles, but it may intensify the defects
of early follicles in high-grade RaRa mice.
We are left now with the possibility that follicle-growth in ragged mice is
retarded directly by an abnormally slow rate of cell-division in the follicle-
forming cells—presumably the epithelial cells of the external sheath. As to the
cause of such a decrease in cell-division rate, there is little direct evidence.
However, an important point is that the follicle-sheath epithelial cells are
indistinguishable cytologically and histologically from the germinative epithelial
cells of the epidermis. These cells make a continuous layer from which small
aggregates of cells form follicle primordia and later develop into diverticuli as
they form follicle external sheaths. There is evidence that these cells, when in
the epidermis of ragged mice, appear to divide normally. First, epidermal thick-
ness, which is related to the speed of cell-division, is always as great as or greater
than normal in Ra+ and RaRa mice. Secondly, the rate of cell-division in
neonatal RaRa mice is not held down to any specific level since it can be arti-
ficially increased by topical applications of testosterone propionate (Slee, un-
published). Thirdly, regions of epidermal hyperplasia, obviously involving high
rates of cell-division, occur in some RaRa mice of all ages. This evidence tells
against the possibility of a general epidermal defect in ragged mice. If we accept
that the epithelial cells of ragged mice can divide at least as fast as normal when
they are situated in the epidermis, but not when they are in hair-follicle external
sheaths, then we must look for physiological differences between these regions
in the control of cell-division. There is some evidence that the unknown stimulus
which initiates the growth of hair follicles (and presumably stimulates cell-
division) is specific to the whole germinative epithelium cell type, whether these
cells are in the epidermis or in the hair follicles (see Chase, 1954; Slee,
1957fl). Therefore, if the follicle sheath and epidermis cells react differently, one
suspects differences at the site of response rather than a failure of one stimulus
at the systemic level. The main difference between epithelial cells of the epider-
mis and of the hair follicles is that the latter become part of a differently organized
structure. As a part of this structure, the dermal papilla may have roles, first as
an organizer and then as a channel for follicle nutrition. Therefore, if it could
be shown that the dermal papillae were absent or defective in many Ra-\- and522 J. SLEE —DEVELOPMENT OF SKIN AND
RaRa follicles, there might be an acceptable explanation for the abnormalities
of follicle development. However, morphological evidence of papilla abnor-
malities is scanty. Some RaRa follicle primordia lacked papillae, especially in
mice affected by oedema, and the cells of these follicles were often diffusely
scattered and disorganized (Plate 2, fig. O). But most RaRa follicles and all
Ra-\- follicles appeared normal in these respects, even those which later became
non-functional, and it seems that if papilla defects are responsible for all
follicle abnormalities of ragged mice they must be mainly undetectable by
ordinary histological procedures. For the present the problem is unresolved,
but some defect specific to the hair follicles themselves seems implicated.
Results of the restrictions in hair-follicle-growth
Many of the obvious effects of the hair-follicle growth retardation have
already been made clear. They can be summarized as follows: (i) failure to
complete development in some follicles, (ii) delay in the completion of develop-
ment in other follicles, (iii) disorganization of the orderly phases of follicle
development which are normally associated with the production of different
fibre types, (i) results in the absence of nearly all fibres in RaRa mice. In Ra-{-
mice, where the late initiated follicles only are affected, many zigzag fibres are
absent, (ii) causes the functional Ra+ hair follicles to be out of phase in their
development and therefore out of phase in their progress through subsequent
hair-cycles, (ii) and (iii) cause hair-structure defects and result in the production
of transitional types of fibre.
In addition, the partial failure of hair-follicle growth may have some effects
on skin thickness. Observations have shown (Text-fig. 1) that the epidermis in
young ragged mice (especially RaRa) tends to be thicker than normal during
the later stages of follicle development. This is strong evidence for a theory
developed by Mottram (1945), Bullough (1952), Chase (1954), and Slee (1957a).
On this theory the epithelial cells of the hair-follicle sheaths and of the germina-
tive layer of the epidermis compete for a substrate supplying energy necessary
for cell-division. So a system of priorities might exist whereby down-growing
hair follicles during the stages of hair proliferation would obtain substrate at
the expense of the epidermal cells. This process could cause the decrease in
epidermal thickness which normally occurs during the advanced stages of
follicle-growth. In juvenile ragged mice, where follicle-growth is slow or arrested,
competition for substrate would be reduced and epidermal cell-division con-
sequently less restricted. It is therefore suggested that the thicker than normal
epidermis generally found in 5-21-day-old ragged mice and the regions of gross
epidermal hyperplasia sometimes seen in RaRa mice result from the restricted
growth of their hair follicles.
The second aspect of skin morphology which seems to be associated with hair-
follicle development is the thickness of the adipose layer. Chase, Montagna, &
Malone (1953) first showed clearly that, in normal adult mice, adiposus thicknessHAIR FOLLICLES IN MICE 523
varied closely with the hair-follicle-growth cycle. Throughout my observations
on adult and juvenile mice of both normal and ragged genotypes it was noticed,
in agreement with Chase et ai, that a thickened adipose layer was always asso-
ciated with hair follicles in the down-growing proliferative stage. Conversely,
a thickened adiposus was never found when the follicles were not in this stage.
This positive association persisted between genotypes and through developmen-
tal stages within genotypes. For example, a comparison of Text-figs. 2 and 5
shows that follicle-growth is clearly related to adipose thickness throughout the
period of juvenile follicle development. Within + + and Ra-\- genotypes adipose
thickness is greatest when follicle length is maximal; in RaRa mice it is always
small, in line with their rudimentary follicle development. Between genotypes
the subnormal adipose thickness of Ra-\- and RaRa mice parallels their sub-
normal follicle development, even to the extent that the delayed retraction in
Ra-\- follicle length with respect to normal is exactly matched by a synchronous
delay in the time of decrease of adipose thickness. If these two phenomena are
associated and synchronous it is simplest to suppose that one causes the other,
rather than to postulate independent causal systems. Therefore it is suggested
that follicle down-growth normally causes expansion of the adipose layer. The
converse, that adipose expansion normally induces follicle down-growth, seems
less likely because in ragged mice many follicles with retarded development
were restricted in size more than could result from restraint due to lack of skin
expansion. Such follicles did not even stretch the full depth of available dermis.
Normally, development of the whole follicle population may cause expansion
of an adipose layer which is refractory, in that a full complement of developing
follicles is required to cause its full expansion. Growth retardation of some
follicles, as in Ra-\- mice, might then be sufficient to make the degree of adipose
expansion subnormal; near agenesis of the follicle population as in RaRa mice
could result in virtual absence of any adipose layer. It is further argued that
the subnormal depth of skin consequently available for those ragged follicles
which do develop fully results in these follicles being constricted. They therefore
become abnormal in shape and orientation (Plates 1 and 2). These morpho-
logical abnormalities seem especially marked after 15 days' post-partum, when
the hair follicles are retracting and skin thickness diminishing. It seems as if the
adiposus normally contracts as retraction of the follicle population permits
it to do so. In Ra-\- mice where the follicles are out of phase in development
some follicles begin to retract before others. Apparently adiposus contraction
commences with retraction of the first follicles, so that the later retracting
follicles, already distorted by their previous growth in a restricted space, become
further compressed by the adiposus contracting ahead of their retractive phase.
Similar anomalies were found in the few large RaRa follicles which occur. Most
of these relationships between subnormal skin depth and abnormal follicle
morphology occur also in adult ragged mice and similar explanations can be
offered there.524 J. SLEE —DEVELOPMENT OF SKIN AND
Control of the mouse hair-cycle
In many rodents hair-growth occurs in a cyclic fashion at regular intervals of
time. Each cycle consists of a period of follicle-growth and hair proliferation
followed by a rest period during which the follicles are quiescent and there is
no hair-growth. A complete cycle lasts 28 days in the mouse. Follicles in any
one region of the body enter the hair-cycle together and follow it in phase with
each other, but follicles in any other region will enter the hair-cycle again in
phase with each other but at a slightly different time from the first region. This
arrangement, by which follicles enter synchronously within body regions but
sequentially between regions upon any specific stage of the hair-cycle, causes
hair-growth waves. Neighbouring hair follicles therefore tend to be in phase
through the hair-cycles, except near the margin of an advancing wave front. The
waves of hair-growth, which are usually bilaterally symmetrical, spread antero-
posteriorly across the body according to a consistent pattern, which has been
well described for the mouse by Dry (1926). The question of how these growth
cycles are initiated and controlled is of fundamental importance and has been
extensively considered, e.g. by Durward & Rudall (1949) and Whiteley (1958).
After a study of adult ragged mice Slee (1957a) concluded that the controlling
mechanism must involve a systemic agency and a degree of follicle autonomy.
There is one particular feature of the ragged syndrome which may be impor-
tant for an understanding of the normal hair-cycle. This is the fact that neigh-
bouring hair follicles can be persistently out of phase with each other throughout
adult hair-cycles, apparently as a result of becoming out of phase during their
initial development in the suckling mouse. This original lack of synchrony
seems to arise by retardation of the later initiated follicles. If this is accepted, it
carries the possible corollary that the synchrony of normal adult hair follicles
within body regions depends upon their becoming in phase during early de-
velopment, where, as we have seen, the late initiated follicles grow sufficiently
rapidly to catch up with those initiated early. Moreover, on this theory, normal
differences between body regions in their time of entry into different phases of the
adult hair-cycles could result from differences in the original time of completing
juvenile follicle development in these regions. This would provide a mechanism
for the hair-growth waves described above. There is not much evidence here for
or against this last possibility, but it is probable that normal follicles on the
anterior dorsum do develop slightly ahead of those on the posterior dorsum.
This fits with the antero-posterior direction of the hair-growth wave found in
subsequent cycles. At this point the evidence of Johnson (1958) from the rat,
and of Nay & Fraser (1954) who used naked mice, must be considered. They
showed that the duration of the hair-growth cycles varied between different
body regions. Developing the ideas of Nay & Fraser we can now see that the
regional differences in hair-cycle duration, which they observed, could interact
with regional differences in the time of cycle commencement to produce wavesHAIR FOLLICLES IN MICE 525 of hair-growth whose pattern varied in successive cycles. Some such changes do occur in the normal mouse (Dry, 1926; Wolbach, 1951). Moreover, if the original wave patterns depended solely upon pre-set synchronies built up during early development one would expect the patterns of later hair generations to become irregular. They do (Fraser & Nay, 1955), and they would not do so if the cycles were self-equilibrating. It is now reasonable to assume that hair follicles are potentially independent in their cyclic behaviour. Although they are normally set off in their development synchronized within body regions, this synchroniza- tion does not seem to be continuously imposed by systemic factors. This is shown by the fact that individual follicles may persistently behave independently of their neighbours due to the action of the ragged gene, or as a result of fibre plucking (David, 1934). It is therefore suggested that synchronization may de- pend entirely upon neighbouring follicles reaching a specific stage of develop- ment at the same time during their original development, and that this degree of synchrony tends to remain for several hair-cycles unless interfered with surgically or genetically. Although some general systemic stimulus may still be required to start each hair-growth cycle, these overall conclusions emphasize the individual hair follicle as an autonomous unit. To some extent this is aetiologi- cally different from the conclusions reached by Ebling & Johnson (1959) after their elegant experiments with the rat. They supposed that the reaction times of different follicles to stimuli initiating a hair-cycle are 'determined by graded local thresholds to a systemic stimulus'. I suggest that follicles' reactions to any systemic stimulus are individually determined and that their times of reaction depend upon their order of completing their original development. Since this order varies between body regions, their times of reaction are in phase within regions and out of phase between regions. If this is accepted, the postulate of graded local thresholds can be discarded. We now have a possible explanation for the form of the rodent hair-cycle, but the question of how the cycles are initiated and terminated remains unanswered. It seems certain that some systemic stimulus is required. Moreover, the relation- ships found between epidermal thickness and follicle-growth in ragged mice reinforce the conclusions of Chase (1954) by indicating that the stimulus does not affect the hair follicles as such but is effective upon a whole cell type. It increases the mitosis rate in at least three components of the germinal epithelium: the epidermal Malpighian layer, the follicle external sheath, and the follicle bulb. There is some evidence that the systemic stimulus may involve a complex endocrine mechanism (Mohn, 1958). Or it can be argued that the removal of a mitosis inhibitor may start each hair-cycle (Chase & Eaton, 1959); this sugges- tion fits with the evidence of Bullough & Laurence (1960), indicating that the removal of an inhibitor may cause an increased mitosis rate in injured epidermis. Whatever the stimulating mechanism, the hair follicles only react to it when they are ready. This moment of reaction for each follicle may depend on its internal physiology becoming responsive at a certain time after the previous cycle.
526 J. SLEE —DEVELOPMENT OF SKIN AND
Obviously there is considerable follicle autonomy, but recent experiments of
Ebling & Johnson (1961) with rats have shown that although individual follicle
reaction times persist to a degree they can slowly be overruled by some form
of systemic stimulus. This was apparent when the two were set at variance by
grafting skin between rats at different stages of the hair-cycle. These results are
not opposed to the view developed above that the reaction time of the individual
follicle is normally set by its original time of development, but they do indicate
that it is not always irrevocably determined. We are left with the concept of an
autonomous follicle response mechanism interacting with a systemic stimulus.
Under certain conditions (e.g. in rat-skin transplants but not, apparently, in Ra-\-
mice) the systemic factor seems able to modify the follicle reaction times.
A complete explanation of the hair-growth cycle is obviously far from being
available, but the question is an important one. The rate of mitosis achieved
by the hair-follicle bulb during the proliferative stage of the cycle is about the
highest achieved by any tissue of the body. Knowledge of how this periodic burst
of cell-division is initiated and so precisely controlled may be fundamental to
an understanding of normal and malignant growth processes.
SUMMARY
1. Hair-follicle initiation and development, and concomitant changes in skin
morphology, were observed in normal ( + + ) mice, and in ragged heterozygotes
(Ra+) and homozygotes (RaRa), from the 14-day embryo until 3 weeks after
birth.
2. Follicle initiation commenced at the normal time (14 days' gestation) in
Ra+ and RaRa embryos. Early initiated Ra+ follicles were normal, but de-
velopment of the later follicles was retarded or arrested. The growth of most
RaRa follicles was arrested early. Grossly retarded follicles (Ra+ and RaRa)
were non-functional and lacked hairs and sebaceous glands. These follicle
defects resulted in lack of zigzag hairs in the adult Ra+ pelage and near-naked-
ness of RaRa adults—as described in a previous paper.
3. The degree of growth retardation in functional Ra+ follicles was variable,
so they became out of phase with each other in their development. This anomaly
was thought to cause in the adult mice intermediate types of fibre and out-of-
phase progress of adjacent follicles through the hair-cycles.
4. Follicle density in Ra-\- mice was subnormal before birth, but near normal
after birth. RaRa follicle density was consistently subnormal. In all genotypes
follicle density was greater on the sacrum than in the neck region.
5. The epidermis in ragged mice became thicker than normal after 5 days
post-partum, possibly because of decreased competition from the hair follicles
for nutrients essential for mitosis. The depth of the adipose layer of the skin
seemed related to the extent of follicle development. It was abnormally thin in
Ra-\- mice and almost absent in RaRa mice. Possibly hair-follicle-growth, here
deficient, is normally required to induce expansion of the adiposus. The shallowYou can also read
