Rogers GEHair follicle differentiation and regulation. Int J Dev Biol 48:163-170

Abstract

Ten years ago, Hardy (1992) wrote a timely review on the major features of hair follicle development and hair growth which she referred to as a secret life. Many of these secrets are now being revealed. The information discussed in this brief review comprises the structure of the hair and hair follicle, the continuing characterisation of the genes for keratin and keratin associated proteins, the determination of the location of their expression in the different cell layers of the hair follicle, molecular signals which control keratin gene expression and post-translational events in the terminal stages of hair formation.

Full text

Hair follicle differentiation and regulation
GEORGE E. ROGERS*
School of Molecular and Biomedical Sciences, University of Adelaide, South Australia
ABSTRACT Ten years ago, Hardy (1992) wrote a timely review on the major features of hair follicle
development and hair growth which she referred to as a secret life. Many of these secrets are now
being revealed. The information discussed in this brief review comprises the structure of the hair
and hair follicle, the continuing characterisation of the genes for keratin and keratin associated
proteins, the determination of the location of their expression in the different cell layers of the hair
follicle, molecular signals which control keratin gene expression and post-translational events in
the terminal stages of hair formation.
KEY WORDS: hair, follicle layer, keratinocyte, keratin gene, gene expression
Int. J. Dev. Biol. 48: 163-170 (2004)
0214-6282/2004/$25.00
© UBC Press
Printed in Spain
www.ijdb.ehu.es
*Address correspondence to: Dr. George Rogers. School of Molecular and Biomedical Sciences, University of Adelaide, South Australia 5005, Australia.
Fax: +61-8-8303-4262. email: george.rogers@adelaide.edu.au
Abbreviations used in this paper: IF, intermediate filament; IRS, inner root
sheath; KAP, keratin associated protein; ORS, outer root sheath.
A hair arises from the integrated activities of several
keratinocyte layers in the hair follicle
Keratinocytes differentiate into several distinct cellular lay-
ers of the follicle during the growth phase (anagen) of the hair
cycle (Figs. 1,2). From the outermost aspect of the follicle the
histological structures are: the outer root sheath (ORS) con-
sisting of several cell layers and the innermost adjacent to the
inner root sheath (IRS) is called the companion layer (Orwin,
1971). Adjoining the ORS on the dermal side is a basket-like
arrangement of two orthogonally arrayed layers of collagen
fibres, the glassy layer (Rogers, 1957) now known as the
dermal sheath.The Henle, Huxley and cuticle layers of the IRS;
the IRS cuticle layer adjoins the cuticle of the presumptive hair
fibre.The presumptive hair shaft comprises an outer layer of
overlapping cuticle cells surrounding a cellular cortex and
sometimes a central medulla.
The primary activities in anagen hair follicle to produce a
hair involve proliferation of the germinative epithelial cells in
the bulb region, the determination of cell lineages for all the
follicle layers (Fig. 2)and terminal differentiation (keratinisation).
The differentiation products of the temporally regulated pro-
cesses consist of structural proteins within the cells and adhe-
sion proteins between the cells that hold them tightly packed
together within the cylindrical-shaped hair.
The region in the bulb where keratinocytes proliferate rap-
idly is called the critical region or hair matrix zone (Auber,
1950, Orwin, 1979) (see Fig. 2); it surrounds the dermal papilla
separated by a basement membrane. Since the seminal ex-
periments of (Cohen, 1961) and (Oliver, 1966) it has been
known that the dermal papilla provides essential stimuli for
both follicle induction and hair growth. The molecular factors
and receptors that are expressed during follicle induction and in
the anagen growth phase are becoming increasingly well-
defined and the fluctuations of their activity are discussed in
reviews (Botchkarev and Kishimoto, 2003, Fuchs
et al
., 2001).
Prominent regulatory proteins in both developing and anagen
follicles include the BMPs, Sonic hedgehog and several WNT
proteins and the receptors, BMPR1A, EGFR, FGFR and TGFR.
A daughter cell from a mitotic event might move out of the
matrix zone of the follicle and differentiate or could remain in the
zone and continue dividing. Whether a cell moves or stays
within the critical region might be controlled by the level of β1-
integrin expression since the expression of β1-integrin is greater
in epidermal stem cells and is reduced when they enter a
differentiation phase (Zhu
et al
., 1999). The importance of β1–
integrin for the maintenance of proliferating cells in the hair bulb
perhaps as a stem cell population was also evident from
observations made on the hair follicles of β1-integrin null mice
(Brakebusch
et al
., 2000). The importance of BMPs in restrict-
ing a normal proliferating population to the bulb region was
demonstrated by the over expression of the BMP inhibitor
Noggin, in transgenic mice. In such mice, dividing cells were
then observed in the hair shaft distal to the bulb (Kulessa
et al
.,
2000).
The synthesis and intracellular deposition of keratin struc-
tural proteins in the spindle-shaped keratinocytes of the cortex
leads to assembly of a composite of intermediate filaments (IFs)

164 G.E. Rogers
and a matrix of keratin associated proteins (called either IFAPs or
KAPs) (Powell and Rogers, 1997), often referred to as the IF-matrix
complex. During keratinisation this composite is finally stabilised
mainly by the formation of inter- and intra-molecular disulfide
bonds. Presumptive cuticle cells that surround the cortex undergo
flattening as they emerge from the bulb region and are interlocked
with the IRS during their passage up the follicle anchoring the hair
in the follicle.
The keratin and keratin-associated proteins expressed
in the cortex and cuticle
The term “keratin” is now generally restricted to designating the
intermediate filament proteins of the fibre cortex. The proteins of
two large families, Type I and Type II, form the IFs of the “hard”
keratins of hair (also the “soft” keratins of the epidermis). The two
keratin families are distinguished by their isoelectric points (Type
I, acidic; Type II, basic/neutral). Equimolar amounts of the two
types are required to form the IFs (Steinert and Roop, 1988).
Studies of the keratins of sheep (Powell and Rogers, 1997)
indicated that the families of chains consist of four Type I chains
and four Type II chains. However, investigation of human hair
keratin gene families have strikingly revealed that the Type I family
has nine members and the Type II has six members (Langbein
et
al
., 1999, Langbein
et al
., 2001). The genes are respectively on
human chromosomes 17q12-21 and 12q13 (Rogers
et al
., 1995).
The keratin-associated (KAP) proteins that constitute the matrix
of the keratin composite of wool are a large group of possibly up to
100 different proteins. Originally, they were divided into three main
families according to their amino acid composition and molecular
size. The proteins containing 35-60 mol% of glycine and tyrosine
(now KAPs 6, 7 & 8, (Powell and Rogers, 1997)) were originally
referred to as the high-glycine/tyrosine group whereas the sulphur-
rich (cysteine-rich) proteins (KAPs 1-5 (Powell and Rogers, 1997))
were divided into a high-sulphur KAP group with less than 30 mol%
of cysteine and an ultra-high sulphur group with cysteine contents
above that value (Gillespie, 1991). Sequence comparisons of the
genes for wool proteins showed that there are at least eight families
of cysteine–rich proteins and two main groups with more than
twenty glycine/tyrosine-rich proteins (Powell and Rogers, 1997).
Studies of the human genome for equivalent genes encoding
glycine-tyrosine rich proteins identified a domain of 17 genes on
chromosome 21q22.1 (Rogers
et al
., 2002). Furthermore, on the
same domain seven KAP genes for high-sulphur proteins were
located which extends an earlier study that revealed a cluster of 37
genes for the sulphur-rich KAP group (Rogers
et al
., 2001) on
chromosome 17q12-2 interestingly within a domain of Type I IF
genes and could be grouped into seven gene families.
The formation of IF and KAP proteins in hair follicles
There is a high rate of protein synthesis in the hair follicle. For
a hair fibre of diameter 100 µm and length growth rate of about 20
µm per h, 5-10µg of protein are produced in a single follicle every
24 h. The large families of
IF
and
IFAP
genes may be necessary
for the high rate of synthesis in enabling transcription of mRNAs at
a level commensurate with demand.
Since the zone of synthesis and maturation in the approximately
lower third of the follicle (called the keratinisation zone) through
which a cortical cell passes is about 1000 µm long, it follows that
the cells are completely filled with the keratin complex and cross-
linked over a period of 48 h as they pass through the zone.
A notable feature is the orderly expression of the IF and KAP
proteins in the developing hair. The temporal sequence with which
they are laid down has been determined by
in situ
hybridisation
detection of specific mRNAs for the different gene families using
cRNA probes (Powell
et al
., 1992, Powell and Rogers, 1997).
These studies (Fig. 3) and a more recent and detailed investigation
Fig. 1 (Left). Light micrograph of the different layers of the hair follicle.
In the
bulb region, a proliferating epithelial matrix surrounds the mesenchymal dermal
papilla. The hair shaft of cortex, medulla and cuticle layers enclosed by the inner
root sheath move outwards within the outer root sheath which is continuous with
the epidermis. (From Millar, 1999; reproduced with permission from Elsevier).
Fig. 2 (Right). Cartoon of the follicle bulb.
The diagram illustrates the several
lineages of cells which are determined in their differentiation pathway and leave
the follicle forming the different layers of the hair and surrounding follicle.

Hair keratinocyte differentiation and regulation 165
(Langbein
et al
., 2001) have conclusively shown that IF genes are
the first to be expressed followed by the high glycine/tyrosine
genes and then the cysteine-rich protein families.
Hair cortex
In situ
hybridisation experiments with cRNA probes (Powell
et
al
., 1992) revealed that IF genes
K2.12, K2.9, K2.10
and
K2.11
for
wool keratins are expressed in that order in the cortical cells
beginning with K2.12 just above the critical zone of the follicle bulb
and the later expression of
K2.11
in the upper bulb region. The
expression is coincident with Type I gene expression. Extensive
studies of human genes (Langbein
et al
., 1999, Langbein
et al
.,
2001) have shown that the pattern of IF gene expression is also
complex and as to which Type I and II proteins out of some twelve
different members specifically pair to form the keratin IFs remains
unclear.
In vitro
experiments, similar in kind to those conducted
with epidermal IFs (Coulombe
et al
., 1990, Coulombe and Fuchs,
1990) to determine the relative affinities of different combinations
of IF proteins would appear to be a major direction for elucidating
the most likely combinations of Type I and Type II chains for hair
keratin IF formation. Such investigations are in progress but so far
inconclusive (Herrling and Sparrow, 1991, Thomas
et al
., 1986,
Wang
et al
., 2000) and an important improvement is the utilisation
of recombinant IF proteins in such recombination experiments
(Hofmann
et al
., 2002).
Ultra-high sulphur KAP families including those of the cuticle are
the last KAPs to be expressed. It should be noted that the
expression of the different gene families are not distinct but merge
one into the other and the translation of their mRNAs continue until
the last stages of hair formation. There is no precise evidence so
far of down-regulation of one set of genes and up-regulation of
another compared to the transcription of epidermal genes (Fuchs
et al
., 1989). Biochemical events are reflected in structural changes
visualised by transmission electron microscopy (TEM) of the
keratinised hair fibre cortex at high resolution. When the cortex is
forming the IFs are seen to be aggregated into fibrils and in
conjunction with the
in situ
expression data it can be concluded that
as the matrix proteins are expressed they migrate into the spaces
within the IF aggregates. The synthesis and insertion of matrix
proteins become coincident processes especially in the late phases
of cortical cell differentiation and aggregation of the IFs. Evidence
for this is that in the keratinising zone the matrix proteins appear to
aggregate as “blocks” (Fig. 4) that subsequently disperse between
the filaments as differentiation advances (Fig. 5). The matrix
proteins and IFs interact further to produce either the typical quasi-
hexagonal or cylindrical packing of the keratinised hair cortex (Fig.
6) (Fraser
et al
., 1972, Rogers, 1959b). The relative abundance of
the two major KAP groups and possibly the IFs as well may be
structural factors responsible for these organisational patterns.
Fig. 3. Localisation of expression of mRNAs encoding the keratin IF
and KAP proteins of wool fibre cortex by
in situ
hybridisation.
Specific
3’-cRNA probes were used to localise the sequential expression of
(A)
keratin IF; (B) Glycine-tyrosine rich KAPs; (C) Cysteine –rich KAPS (< 30
mol%) and (D) Cysteine-rich KAPs ( >30 mol%), the last to be expressed.
Fig. 4 (Left). Electron microscopic image (TEM) of loose bundles of
keratin IFs in a longitudinal section of a wool follicle.
The TEM shows
the presence of dense aggregates (arrow) within bundles of IFs (macrofibrils)
and are assumed to be KAP proteins. They are also readily observed in
cross sections at a later stage of follicles, in which it can be seen that the
granules begin to disperse and interact with the IFs (see Fig. 5).
Fig. 5 (Right). TEM of part of a cortical cell in an osmium-fixed follicle.
The aggregates seen as “blocks” in Fig. 4 appear as electron-dense matrix
incompletely distributed between the IFs which constitute macrofibrils. Dense cytoplasmic material is abundant between the macrofibrils. This
differentiation process occurs in cortical cells at mid level of the keratinisation zone of a hair follicle.
A
BC
D

166 G.E. Rogers
From X-ray diffraction studies and TEM studies (Jones
et al
.,
1997, Strelkov
et al
., 2003) it has been deduced that the
organisation of keratin chains within the IFs consists of a regular
array of dimer units and an average of 32 chains in the IF cross-
section. However the precise location of covalent links between
IFs and especially between the IFs and matrix proteins have yet
to be mapped in a detail that would more precisely explain the
physical properties of hair.
Hair cuticle
The amorphous cystine-rich contents of scale cells (Bradbury,
1973, Fraser
et al
., 1972) consist of at least two unique families of
proteins (KAP5 and KAP10) of the cuticle (Fig. 7) (Jenkins and
Powell, 1994, MacKinnon
et al
., 1990, Rogers
et al
., 2004). The
evidence for the expression of specific IF proteins, Type I (hHa2
and hHa5) and Type II (hHb2 and hHb5) in the developing cells was
unexpected (Langbein
et al
., 2001, Rogers
et al
., 1996). These
chemical findings appear to be in conflict with the microscopic
evidence from TEM that IFs are not prevalent in developing cuticle
cells. Some “tufts” of IFs can be visualised in the developing scale
cells in the bulb region but the prominent structures that are
produced are globular masses that fuse to form the exocuticle
(Orwin, 1979, Powell and Rogers, 1997, Rogers, 1959a, Rogers,
1959b). The KAP 5 and KAP10 proteins are major components of
the exocuticle (unpublished observations). An explanation might
be that the IF proteins are degraded in the later phases of
differentiation. Other proteins yet to be confirmed in scale cells are
those related to keratinocyte cell envelopes such as involucrin and
loricrin (Kalinin
et al
., 2002, Steinert and Marekov, 1995) that are
cross-linked by isopeptide bonds (Rice
et al
., 1994). An additional
feature of the hair cuticle is the presence of a group of long-chain
fatty acids that is responsible for the hydrophobicity of the hair
surface. Chemical evidence indicates that the fatty acids are linked
by thioester bonds to protein(s) that are probably components of
the scale cell envelope (Jones and Rivett, 1997). Unexpectedly
more than 50% of the fatty acids is 18-methyleicosanoic acid
(MEA) and a genetic defect affecting the synthesis of MEA pro-
duces structural defects in the intercellular layers of the cuticle. The
details of the site and synthesis of these cuticular proteolipids are
unknown. A summary of the structure of the cuticle of hair in shown
in Fig. 8.
Inner root sheath
The IRS is adjacent and adherent to the growing hair and is
responsible for the surface topography of the fibre. It is degraded
and sloughed as the hair emerges from the follicle. The establish-
ment of the IRS cell lineage is dependent on the activities of several
factors and recently the transcription factor GATA-3 has been
identified as playing a central role. The IRS is not formed in
GATA-
3
null mice, the normal coat is absent and aberrant hairs are
produced from embryonic skin grafted onto nude mice (Kaufman
et al
., 2003, Kobielak
et al
., 2003).
The IFs in the IRS cells are morphologically indistinguishable
from IFs of the cortex but markedly different in protein composition.
The expressed genes for a Type I in sheep have been identified
and localised in the IRS (Fig. 9), one Type II in mice (Aoki
et al
.,
2001) and four in human (follicles) (Langbein
et al
., 2003). It is not
known whether they specifically pair but the deficiency of one IF in
the IRS through a spontaneous mutation in mice, causes collapse
of the IF network and interrupts normal IRS formation (Peters
et al
.,
2003). It would seem likely that there are more members of the IRS
intermediate filament family to be found.
Differentiation of the IRS is characterised by the synthesis of
trichohyalin a specific precursor matrix material that finally binds
the filaments into a cross-linked composite analogous to that of the
cortex. As the cells move upward with the hair the trichohyalin that
is present as cytoplasmic aggregates, undergoes a post-transla-
tional modification of arginine residues to citrulline causing the
protein to disperse between the filaments (Rogers
et al
., 1997,
Rothnagel and Rogers, 1986). The final IF-matrix composite does
Fig. 6 (Left). Cross-section of
macrofibrils of the developing cortex at
a late stage of differentiation.
The kera-
tin IF in the macrofibrils are completely
separated by the electron-dense matrix
consisting of the KAP proteins which have
dispersed between them. The IFs display
the cylindrical mode of packing. The cyto-
plasmic material between the macrofibrils
has markedly decreased in abundance.
Fig. 7 (Right). Localisation of the ex-
pression of
KAP 5
mRNA by
in situ
hybridisation.
The expression of the
KAP
5
gene was detected using a 3’- cRNA
probe. Strong expression signal appears in
the cuticle at a late stage of hair formation
in a human follicle.

Hair keratinocyte differentiation and regulation 167
not have the quasi-crystalline packing seen in cortical keratinocytes
and instead of disulphide bonds it is extensively cross-linked by
isopeptide bonds produced by transglutaminase activity present in
the follicle.
Other differentiation products reported in the cells of the IRS
include µ-crystallin, a protein primarily found in the eye. It pos-
sesses both enzymic and structural properties (Aoki
et al
., 2000).
Several proteins are calcium-binding proteins, namely trichohyalin
itself and the enzymes, peptidylarginine deiminase and
transglutaminase
Why are the proteins of the IRS different from those of the hair
shaft? The answer surely lies in the requirement for IRS cells to be
finally sloughed as the anagen hair emerges from the skin surface
and is released from the supporting layers of the IRS. This change
is achieved by the proteins of the IRS being readily degradable by
proteases (Rogers, 1964a) whereas the molecular organization of
hair keratin makes it resistant to proteolysis. Proteolytic activity has
been observed to be a central feature of epithelial desquamation
and is present in the hair follicle distal to the opening of the
sebaceous duct (Ekholm and Egelrud, 1998).
Outer root sheath
The outer root sheath (ORS) is continuous with the epidermis
but the layer of cells immediately adjacent to the IRS Henle layer
has some features that differentiate it as a distinct entity (Rogers,
1964b)]. Notable are tufts of intermediate filaments located at the
ORS/IRS junction complex oriented so that they encircle the
follicle. This layer was later named the companion layer (Orwin,
1971) but it’s role in the dynamics of hair follicle function is unclear.
Certainly the junction adjoining the Henle layer is different in
lacking desmosomes and that could indicate that the IRS moves
relative to the companion layer during outward growth of the hair.
The alternative is for the layer to move with the IRS as that layer
differentiates with the growing hair. Families of K6 proteins to-
gether with K16 (Rothnagel and Roop, 1995, Takahashi
et al
.,
Fig. 8 (Left). Diagrammatic representation of a scale cell of the hair cuticle.
The major
intracellular layers, hydrophobic layer, A layer, exocuticle and endocuticleand protein components
of a scale cell are indicated in cross-section.
Fig. 9 (Right). Localisation by
in situ
hybridisation of the expression of the mRNA encoding
a Type I IF protein.
The mRNA detected by a 3’- probe is highly expressed in the IRS of a hair follicle.
The dark material in the bulb region is melanin.
1995, Winter
et al
., 1998) are expressed in the companion layer
and presumably they correspond to the keratin IFs seen in the cells.
Signals which regulate cell specificity and gene expres-
sion
The regulatory molecules and their networks that control differ-
entiation of hair keratinocytes are becoming increasingly defined
and indeed complex. Notch has been identified as a factor in the
determination of cell type (Kopan and Weintraub, 1993, Lin
et al
.,
2000). BMP signalling inhibits follicle development (Botchkarev,
2003) and when the abundance of BMPs was reduced by over-
expressing Noggin the differentiation of keratinocytes into mature
cortical and cuticle cells was severely impaired demonstrating the
key role of BMPs in the formation of the hair layers (Kulessa
et al
.,
2000). The central role of BMP and its linkage to the WNT pathway
has been strikingly substantiated (Kobielak
et al
., 2003) by knock-
ing out the gene for the BMP receptor BMPRIA resulting in hairless
mice with malformed follicles. Follicle growth was inhibited and the
follicles lacked an IRS although those features are not necessarily
causally related.
The regulation of gene activity in the anagen hair follicle shares
a significant degree of commonality with the keratinocytes of the
epidermis for which a large number of transcription factors for
both positive and negative regulation have been recognised
(Eckert
et al
., 1997, Fuchs
et al
., 2001, Nakamura
et al
., 2001).
The involvement of LEF1 in hair keratin gene expression indi-
cated the participation of this factor in hair follicle development
through the WNT regulatory pathway (DasGupta and Fuchs,
1999). The importance of that pathway has been demonstrated by
numerous findings that when it is dysfunctional through natural or
experimental mutations, hair follicle development is affected and
produces a variety of hair and follicle phenotypes. It is also
essential for the maintenance of inducing activity of the dermal
papilla (Kishimoto
et al.,
2000; Shimizu and Morgan, 2004). The
central molecule in the WNT pathway is β-catenin which has dual

168 G.E. Rogers
roles of being part of the cadherin complexes of cell junctions or
acting within the nuclei of cells after transportation from the
cytoplasm (DasGupta and Fuchs, 1999, Merrill
et al
., 2001) and
forming a transcription complex with the LEF1/TCF DNA binding
family of proteins. This complex activates genes involved in hair
follicle development and presumably, hair keratin genes as well,
given that the
LEF1
consensus is present in the proximal promot-
ers referred to earlier.
An early survey for control sequences in the 5’ promoter region
of several hair keratin genes (Powell
et al
., 1992, Powell
et al
., 1991)
revealed several binding sites that are commonly active in the control
of gene expression. A sequence
CTTTGAAGA
was found to be
common to some 15 hair keratin genes and located between 180 and
240 bp upstream of the transcription start sites (Powell
et al
., 1991).
This sequence was later recognised as the site for LEF1 binding
(Zhou
et al
., 1995) An investigation using a
K2.10 –lacZ
transgene
expressing in transgenic mice, demonstrated that all the regulatory
elements for expression appear to be located in 400 bp of the
promoter of the
K2.10
gene (Dunn
et al
., 1998). Reduction of the
promoter to 200 bp including deletion of the LEF1 site resulted in no
expression of the transgene. Site-directed mutagenesis of the LEF1
binding site in the 400 bp transgene allowed patchy expression and
indicates that a different factor(s) is required for follicle cell specificity.
The presence of trans-acting regulatory factors that bind to the
promoter of the
K2.10
keratin gene was demonstrated in hair follicle
extracts by DNAse-1 foot printing.
It has been suggested (Powell and Beltrame, 1994) that the
coordination of keratin gene expression could be under the control
of locus control regions (
LCRs
) that open up chromatin domains
and thereby direct the activation of keratin genes as found for the
globin gene loci (Trimborn
et al
., 1999). The regions for control of
expression would include Type I and Type II gene families of hair
and epidermis keratin IF linked into separate large clusters (Powell
and Rogers, 1997) and
KAP
genes that are linked to the Type I
keratin
IF
domain an organisation that has been more recently
extensively revealed (Langbein
et al
., 1999, Langbein
et al
., 2001,
Rogers
et al
., 2004). At the present time it is presumed that the
establishment of loci of different families of homologous genes
arose by gene duplication but the elements controlling the activity
of these loci have yet to be elucidated.
The final events of keratinisation
As the regulated expression of the keratin genes progresses to
the last stages and the IF and KAP proteins are laid down, the
keratinocytes of the differentiating hair and the accompanying IRS
cells undergo other structural changes.
Desmosomes, gap junctions and tight junctions are established
between differentiating keratinocytes of the hair fibre and the IRS
to varying extents on their upward journey in the follicle. According
to electron microscopic studies (Orwin
et al
., 1973a, Orwin
et al
.,
1973b, Orwin
et al
., 1973c) gap junctions and desmosomes cover
about 10% of the plasma membrane surface of cortical cells in the
bulb region and then gradually degenerate. Desmosomes are
more abundant in the IRS compared to the developing cortex. Tight
junctions (zonula occludens) are also established between Henle
and Huxley layers and between Henle cells and those of the
apposed companion layer of the outer root sheath (ORS). These
junctions probably alter the movement of small molecules (signal-
ling molecules and metabolites) between the cells.
In the differentiation of both hair and the IRS keratinocytes, the
junctions are replaced with a new cell membrane complex (CMC)
that gradually develops as a continuous layer between the cells.
This complex consists of an electron-dense central (δ) layer about
15nm thick surrounded by β-layers that are approximately 5nm
wide (Jones and Rivett, 1997, Rogers, 1959a, Rogers, 1959b).
Once the growing hair has passed through the keratinisation zone
in the lower third of the follicle, morphological changes occur in the
nuclei and cytoplasm of all the cells. Although nuclei remain in the
cells the chromatin is degraded and mostly resorbed. Several
markers have shown that apoptosis participates in the mecha-
nisms that occur during morphogenesis of the hair follicle and in
catagen and anagen of the hair cycle (Magerl
et al
., 2001, Müller-
Röver
et al
., 1998). A low degree of apoptosis continues in the outer
root sheath of the anagen follicle but not in the keratinising hair
shaft above the bulb region. Instead the nuclear membrane be-
comes insoluble and remains in the keratinised cell as an elon-
gated structure in the cortex. The changes that make it insoluble
are probably isopeptide links (Rice
et al
., 1994).
The aqueous milieu that supports the biochemical processes of
differentiation disappears in the late stages of hardening. The loss of
water is probably aided by the rapid disulfide cross-linking of sulfhy-
dryl groups in the newly synthesised keratin proteins (1650 µmoles
cysteine/g reduces to about 30 µmoles/g; see Gillespie (1991). This
event occurs at the upper region of the keratinisation zone over a
distance approximating the length (100 µm) of a cortical cell. How
these events are catalysed and regulated is not known although
copper in some biochemical form has been implicated in wool growth
(Gillespie, 1991, Marston, 1946, Marston, 1949).
Acknowledgements
Dr. Lesley Jones kindly provided the TEMs for Figs. 5 & 6 and I am
grateful to Dr. Barry Powell for helpful discussions.
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