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Short Communications |
From the Departments of Dermatology*
and Genetics
and Development,
Columbia University, New
York, New York; and the Department of
Dermatology,
University Hospital Eppendorf,
University of Hamburg, Hamburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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During the last decade, it has been shown that the expression patterns of many cytokines, transcription factors, and adhesion molecules are subject to significant changes during normal HF morphogenesis and cycling8,9 thus suggesting a role in the regulation of HF transformation. Although informative, these studies are somewhat limited in that they do not illuminate the molecular pathways of gene activity and their functional role in HF biology. In contrast, expression studies may serve as a valuable extension to the molecular and genetic studies and provide substantial insights into the mechanisms of HF functioning, because they can identify the localization and immediate cellular targets of particular regulatory molecules. Therefore, the combination of genetic and functional analysis of mutant mouse models with expression studies in normal mouse skin is among the most powerful experimental approaches in hair research.
The hairless (hr) gene, which encodes for a putative zinc finger transcription factor10 is one of the candidate genes for the regulation of basic HF functions.6 This gene is the target of several allelic mutations in laboratory rodents,11-13 humans,14-16 and monkeys.17 The attenuation of hairless gene activity in hr/hr mutants results in the progressive shedding of the infantile hairs in animals, which represents an analog of the autosomal recessive disorder papular atrichia (MIM 209500) in humans.14,18 In addition to HF abnormalities, homozygous hr/hr mouse mutants display immunological skin dysfunction, elevated sensitivity to UV and chemically induced skin carcinogenesis,19,20 a unique susceptibility to dioxin skin toxicity,21 and structural abnormalities in the inner ear, retina, and colon,13 thus suggesting possible pleiotropic effects of hr gene mutations in humans as well.18
Our recent studies on the successive pathomorphology of hairlessness have revealed that the attenuation of hr gene activity results in premature and excessive apoptosis and discoordination of cell death, proliferation, and adhesion in selected HF cell populations during the first transition from the anagen to the catagen phase of the HF cycle.6,22 These functional and morphological studies were supported by recent progress in understanding molecular aspects of hr gene biology.15,23 At the same time, however, some basic questions surrounding hr gene biology remain unanswered. For example, does the expression of the hr gene co-localize to the sites of the main pathomorphological events that underlie the process of hair shedding in mutant mice? Are the cellular structures involved in the pathogenesis of the hairless phenotype direct targets of hr gene activity? What are the temporal patterns of hr gene expression in skin and which stages of HF transformation are associated with minimal and maximal gene expression? Does the hr gene serve as a regulator of HF progression during a certain stage of the cycle or is hr a key factor in modulating the transition from one stage of hair cycle to the next—a stage-switch factor?
In an initial effort to address these questions, we have used nonradioactive in situ hybridization to localize and characterize hr gene expression throughout HF morphogenesis and cycling in normal mouse skin. The combination of expression data presented here, together with our previous functional studies, allows us to posit that the hr protein is directly involved in the coordination of cell proliferation and cell death, in particular, in epithelial cell populations during HF catagen progression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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C57BL/6J mice with normal HF cycling behavior were purchased from Jackson Laboratory, Bar Harbor, ME.
For studies of HF morphogenesis, newborn pups (0, 3, 5, 12, and 18 days postpartum) were used. For synchronization of the hair cycle, 8-week-old female mice with all back skin HFs in telogen were depilated with a wax and rosin mixture.24 Animals were sacrificed by CO2 asphyxiation at defined stages of the HF cycle (days 1, 3, 5, 12, and 18 after depilation corresponding to telogen; anagen II, IV, VI; and catagen, respectively). Three mice were studied at every stage of HF morphogenesis and cycling. The perfusion of mice was performed with ice-cold phosphate-buffered saline (PBS) (Gibco BRL, Grand Island, NY) and 4% paraformaldehyde (EM Science, Gibbstown, NJ) in accordance with standard protocols.25 The dorsal skin samples were fixed in cold 4% paraformaldehyde overnight, washed in PBS, and embedded in paraffin according to standard procedures. Five-µm sections were mounted on silane-coated glass slides (six slides per mouse with three sections each) and prepared for in situ hybridization as previously described.26
The stages of HF morphogenesis were assessed according to Hardy’s classification27 with modifications suggested by Philpott and Paus.2 The stages of HF cycle were assessed according to Paus et al.28 On every section, five to fifteen well-sectioned HFs were analyzed.
Probes
The hr cDNA fragment was obtained by polymerase chain reaction using primers spanning nucleotides 3212 to 3233 and 3657 to 3678 (GenBank accession number Z32657) and mouse skin cDNA as a template. The fragment was ligated into a pCRII-TOPO vector (Invitrogen, Carlsbad, CA), and propagated using One Shot TOP10 competent cells (Invitrogen, Carlsbad, CA). The clones encoding the specific cDNA were identified by direct sequencing on both strands using ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).
Digoxigenin-labeled sense and antisense riboprobes were produced in vitro using Sp6 and T7 RNA polymerases (DIG RNA Labeling Kit; Boehringer-Mannheim, Mannheim, Germany) according to the manufacturer’s protocol. The mouse keratin 1 (MK1) riboprobe was obtained as described.21
In Situ Hybridization
In situ hybridization was performed as previously described.26,29 Briefly, deparaffinized skin sections were acetylated in acetic anhydride solution (EM Science) and then dehydrated. Hybridization with 50 ng/section of freshly denatured cRNA probes was performed at 50°C for 17 hours in the humidified chambers. The mouse hr sense probes were used as a negative control, and the mouse keratin 1 antisense probe as a positive control. Incubation with sheep alkaline phosphatase-labeled anti-digoxigenin antibodies (DIG Nucleic Acid Detection Kit, Boehringer-Mannheim) was performed for 3 hours in humidified chambers at room temperature. Some control slides were incubated in the absence of antibodies. Then the slides were stained by incubation in nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate solution (Boehringer-Mannheim) for 16 to 20 hours in complete darkness at room temperature.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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In the epidermis of neonatal mouse skin (day 1 postpartum),
hr mRNA reactivity was present in the suprabasal cell layers
with a gradual decrease of intensity in the spinous compartment,
concomitant with keratinocyte differentiation (Figure 1A)
. During the initiation of HF
morphogenesis, no hr mRNA immunoreactivity was found in the
epidermal placode nor in the dermal fibroblast condensation (not
shown).
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, similar to hr expression patterns
in the interfollicular epidermis. These hr mRNA-positive
follicular keratinocytes remained in the upper, epidermis-associated
portion of the follicle that corresponds to the infundibulum of the
mature HF (zone 1; Figure 1, B and C
). This pattern of hr
expression in the infundibulum persists throughout HF morphogenesis.
The lower, downward-growing portion of the HF peg remained
hr-negative at the earliest stages of HF morphogenesis.
Later, at stage 4 of HF morphogenesis, along with formation of the hair
matrix, the hr mRNA-positive staining appeared (Figure 1B)
and progressively increased (Figure 1C)
in the lower HF portion (zone 2
of expression). During the formation of the precortex and cone-shaped
inner root sheath (IRS), and consequent initiation and progression of
hair shaft growth, strong hr mRNA positivity was observed in
IRS keratinocytes (zone 3; Figure 1, C and D
).
Thus, in addition to the consistent expression in the
interfollicular epidermis, three zones of cell type-specific expression
of hr gene were established during HF morphogenesis: in the
HF infundibulum (zone 1), in the hair matrix (zone 2), and in the IRS
(zone 3). No expression was observed in the lower and middle outer root
sheath (ORS) or the dermal papilla (DP) fibroblasts throughout the
entire process of HF morphogenesis (Figure 1, D and E)
.
hr Gene Expression during Depilation-Induced HF Cycling
Adolescent mouse skin displayed consistent hr
expression in the suprabasal cell layers of the interfollicular
epidermis. The suprabasal keratinocytes of the HF infundibulum were
also consistently hr mRNA-positive during the entire HF
cycle (Figure 1J)
.
In contrast to the epidermis and HF infundibulum, the expression patterns of hr mRNA in the lower portion of the HF epithelia were strictly hair cycle-dependent, and are summarized below.
Telogen
In the telogen or resting phase of HF cycling, prominent
hr mRNA immunoreactivity was found in the cells localized to
the lowermost portion of the follicle bulb in contact with the DP (the
zone of the secondary follicle germ). Some other keratinocytes of the
HF, primarily localized to the lower portion of the bulb, were slightly
or moderately hr mRNA-positive as well (Figure 1F)
.
Anagen
During the telogen-anagen transition, the hr
mRNA-positive keratinocytes of the lower portion of the HF moved
downward (Figure 1G)
, and in anagen III, gave rise to prominent
hr gene expression in keratinocytes of the HF matrix (Figure 1H)
and Huxley’s layer of the IRS, excluding the completely cornified
IRS portion (Figure 1I)
. During the advanced stages of anagen HF
development, hr mRNA immunoreactivity was also localized to
the thin innermost cell layer of the upper ORS (Figure 1J
, arrowhead).
The sebaceous gland cells and keratinocytes of the bulge region were
consistently hr mRNA-negative (Figure 1J
a).
Catagen
The most prominent changes in hr mRNA expression
patterns were observed during the catagen phase of HF transformation.
The spontaneous switch from depilation-induced anagen to catagen was
associated with a rapid decline of hr mRNA immunoreactivity
in the HF matrix (Figure 2,A
and B). hr mRNA-positivity in IRS
keratinocytes during the initial and mid-catagen phases (catagen I to
V) remained high (Figure 2, B and C)
, but declined along with
progressive IRS cornification and disintegration in catagen VI to VII
(Figure 2, D and E)
.
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, black arrowhead). At
that point, the club hair begins to form by means of trichilemmal
keratinization of ORS keratinocytes and their interdigitation with the
cells of the hair shaft.30
At the next stage of catagen
(VI), a significant increase of positive staining in this zone was
observed (Figure 2D)
. The completion of club hair formation in catagen
VII was associated with a rapid decline of hr mRNA
expression and its complete disappearance during HF progression into
telogen (Figure 2E)
.
In late catagen, hr mRNA was localized in a few cells of the
regressing epithelial strand just above the DP (Figure 2D
, white
arrowhead). The epithelial strand contraction and upward movement
of the DP during catagen VII to VIII was associated with an increase in
hr mRNA expression in this particular keratinocyte
population. This population also moved upward and apparently provided a
source of hr-positive keratinocytes in the telogen HF
(Figure 2, D–F)
. These DP-associated epithelial cells remained highly
hr mRNA-positive throughout the entire telogen stage (Figure 1F)
.
MK1 Expression in C57BL/6 Mouse Skin
In situ hybridization with a digoxigenin-labeled
riboprobe specific for MK1 was used as positive control. The positive
MK1 mRNA staining was clearly seen in the suprabasal cell layers of the
interfollicular epidermis as previously reported by Schweizer et
al31
(Figure 2H)
. This MK1 expression pattern along with
the results of application of the sense hr riboprobe (Figure 2G
; negative control) confirms the specificity and sensitivity of our
in situ hybridization technique. The slides processed in the
absence of anti-digoxigenin alkaline phosphatase-labeled antibodies
displayed no staining after the final stage of detection (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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hr Expression in the Interfollicular Epidermis Is Stable
The expression patterns of the hr gene in the suprabasal keratinocytes of the interfollicular epidermis and in the suprabasal keratinocytes of the HF infundibulum, which is normally characterized by epidermal patterns of keratinization,32 were found to be identical. These findings suggest that the characteristic dilation of utriculi in the skin of mutant hairless mice reflects hr-related abnormalities in the interfollicular epidermis rather than in the HF itself. The interfollicular epidermis and utricular epithelium in hairless HRS/J hr/hr mutant mice are characterized by excessive cornification.22,33 This feature is associated with simultaneous up-regulation of both keratinocyte proliferation22 and apoptosis,34 suggesting an elevated rate of cell turnover in the epidermis and utricular epithelium of hairless mouse skin. Because hr is actively expressed in both of these cell populations (in normal interfollicular epidermis and in the HF infundibulum, which corresponds to the utricular epithelium), it might be implicated in the regulation of the balance of cell proliferation and terminal differentiation in selected epithelial cells.
hr Gene Is not Involved in the Initiation of HF Morphogenesis
During the earliest stages of HF morphogenesis (stages 0 to 2), hr mRNA immunoreactivity was observed in neither the epidermal placode nor in the dermal fibroblast condensation and the DP was consistently hr mRNA-negative, thus essentially excluding hr from among the regulatory factors involved in the initiation of HF morphogenesis. This observation is consistent with the entirely normal development of the first pelage hairs in homozygous hr/hr hairless mutants until the onset of first catagen.6
hr Expression in the Anagen-Catagen Transition
The initial stages of catagen in normal C57BL mouse skin are
associated with a decline of hr mRNA immunoreactivity in the
keratinocytes of the hair matrix (Figure 3)
. As we have shown previously, in
hairless mouse skin the switch from anagen to catagen is associated
with a dramatic and premature up-regulation of apoptosis in the HF
matrix.6
These observations, together with data on the
discoordination of keratinocyte apoptosis and differentiation in
epidermis and utricular epithelium of hairless mouse
skin,22,34
suggest that normal expression of the
hr gene seems to regulate the balance between apoptosis and
differentiation in selected HF keratinocyte populations.
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.6
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The onset of club hair formation was associated with a rapid
increase in hr gene expression in the ORS cells surrounding
the zone of interdigitation between the ORS keratinocytes and the hair
shaft cells. As soon as the club hair is formed, hr gene
expression in this region rapidly declined (Figure 3)
. In hairless
mouse skin, the normal club hair does not form, and instead it turns
into an amorphous bulbous structure, in contrast to the serrated club
hair in normal skin (Figure 4)
.6
It is widely accepted
that trichilemmal keratinization is the main mechanism of club hair
formation.30
The possible involvement of hr in
the regulation of trichilemmal keratinization is also supported by its
elevated expression in a spatially restricted portion of the isthmus
ORS between the upper end of the IRS and the sebaceous gland duct,
which is also characterized by formation of a specific keratinous comb
because of trichilemmal keratinization of ORS
keratinocytes.35
hr Gene May Be Involved in Upward Movement of the DP
In late catagen, a small cluster of epithelial strand cells
adjacent to the DP exhibits a substantial level of hr mRNA
positivity. Other cells of the epithelial strand are weakly positive or
negative (Figure 3)
. Recently, we proposed that the keratinocytes in
this particular zone of the epithelial strand may be implicated in the
continuity of DP-epithelial integrity in the catagen HF.6
The hr gene expression in these cells also supports this
notion. In hairless mutants, the loss of hr gene activity in
this particular cell cluster may be the reason for the loss of
integrity between DP and the rest of the ascending epithelial
strand. These findings, along with the absence of hr
expression in the DP throughout all stages of HF cycling, suggest that
the mechanism of DP separation in hr skin and the subsequent
failure of HF cycling is not intrinsic to the DP itself, but rather to
its epithelial mooring.
Major Defects in Hairless Skin Coincide with hr Expression
Our previous studies of hairless mouse skin during the initiation
and progression of hair loss (days 14 to 21 postpartum) revealed
several characteristic abnormalities.6
First, the
dysregulation of cell proliferation and death in the HF matrix resulted
in mispositioning of the ORS, IRS, and hair shaft (Figure 4)
. Second,
the club hair is excessively large and lacks its normal serrated
appearance. Third, the secondary HF germ fails to form. Fourth, the
epithelial strand in hr/hr skin is not able to undergo its
normal contraction, and instead disintegrates into separate cell
clusters. Finally, the DP fibroblasts remain stranded in the dermis,
surrounded by a few epithelial cells. As shown in this study, the
temporal and spatial patterns of hr gene expression in the
normally haired skin of C57BL mice coincide with most morphological
defects in hairless mutant skin, thus implicating the hr
gene as one of the key factors in coordinating basic cellular processes
during HF catagen, including club hair formation, maintenance of
DP-epithelial integrity, IRS disintegration, and keratinocyte apoptosis
in the HF matrix.
At the same time, the diminution of hr gene activity in the matrix during the HF morphogenesis does not result in any apparent abnormalities. In normally haired skin of C57BL mice, hr is actively expressed in the matrix keratinocytes during first postnatal anagen hair growth. In hairless and rhino mice with reduced or absent activity of this gene, HF morphogenesis is apparently normal. Perhaps in this particular zone of HF epithelia, hr may share functional redundancy with other genes, and hr gene functions are not critical for the support of matrix cell homeostasis during HF anagen development.
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Conclusions |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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Collectively, our findings provide new insights into the pathobiology of the hr mutation, and suggest that the normal hr gene product is involved in the spatial and temporal coordinating of the expression of genes required for regulation of cell proliferation, differentiation, and death which together maintain the normal tissue architecture of the HF during the catagen progression.
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Acknowledgements |
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Footnotes |
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Supported in part by grants from the Dermatology Foundation (to A. A. P.), the National Alopecia Areata Foundation (A. M. C.), and from Wella AG and Deutsche Forschungsgemeinschaft (Pa 345/8–1; to R. P.).
Accepted for publication June 26, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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