Epidermal morphogenesis and analysis of the murine skin

The skin is a multilayered tissue which protects the animal from loss of water, bacterial infections, radiation, extreme temperatures and mechanical stress. It is composed of more

interfollicular epidermis), the hair follicle (HF) and as well as a mesenchymal compartment consisting of dermis, subcutis and dermal papillae. The major cell type of the epidermis is the keratinocyte.

Targeted ablation β1 integrins from basal keratinocytes demonstrated the essential of role β1 integrins for the maintenance of epidermis and HFs (Brakebusch et al. 2000; Raghavan et al.

2000). In the present study, the role of ILK in the epidermis and its appendages was addressed. The following sections will introduce the development of the skin and HFs and discuss the role of ECM-integrin interactions in the epidermis.

1.6.1. Epidermal morphogenesis

The epidermis of mice derives from the outer ectodermal cell layer of the postgastrulation embryo that forms a single sheet of histologically undifferentiated epithelial cells which adhere to an underlying BM (Fig 1.19A). Already at E9-E12 these cells regionally stratify to form the periderm, a cell layer which later during epidermal development is shed into the amniotic fluid (Fig 1.19B). Further stratification leads to the formation of a first intermediate cell layer (also called the stratum intermedium) which contains still proliferating cells (Fig 1.19C). Around this time, the expression of the typical keratinocyte marker such as keratin5 (K5) or keratin14 (K14) is induced. Further differentiation at E15-E16 gives rise to the non-proliferating suprabasal cell layers (Fig 1.19D). Terminal differentiation of suprabasal cell layers leads to the formation of the outer epidermal layer called stratum corneum and the shedding of the periderm (Fig 1.19E). The stratum corneum consists of anucleated and flattened cells, which are filled with keratin matrix and surrounded by an impermeable cornified envelope that is additionally cross-linked to external lipids. The stratification of the epidermis is completed at birth (Fig 1.19F; Blanpain and Fuchs 2006).

The epidermis constantly renews itself throughout the entire life of the animal and is able to re-epithelialize after wound injuries, which implies the existence of epithelial stem cells (eSC). Long-term labelling studies revealed that most of those eSCs are located in the hair bulge which is located at the base of the permanent part of the HF (Cotsarelis et al. 1990).

There is also evidence for the existence of eSC in the interfollicular epidermis as well (Mackenzie 1997) and it has been speculated that high β1 integrin expression is a hallmark of eSC (Jones et al. 1995). However, to which extend β1integrin determines the features of eSC awaits further studies.

Fig 1.19. Development of the murine epidermis. A. The epidermis derives from an ectodermal cell layer of the postgastrulation embryo. B. The first stratification occurs around E9-E12 and forms the periderm, which sheds during embryonic development into the amniotic fluid. C. The stratum intermedium is formed between E12-E15 but is not regarded as a typical suprabasal cell layer, since the intermediate cells express basal markers and still proliferate. D. Further differentiation leads to the formation of suprabasal non-proliferating cell layers around E15-E16. E. The formation of the granular layer occurs at E16-E17. The periderm is shed into the amniotic fluid. F. The stratum corneum forms until birth by terminal differentiation. (Based on Blanpain and Fuchs 2006).

1.6.2. HF morphogenesis and the hair cycle

The HF is an epidermal appendage that starts to form already during embryonic development (Fig 1.20). Undifferentiated ectodermal cells are induced by the underlying mesenchymal cells to form an epidermal placode (Fig 1.20B), which in turn induces the formation of a dermal condensate that develops into the dermal papilla (DP; Fig 1.20C). Signals from the DP stimulate the proliferation and differentiation of epidermal cells resulting in the formation of the epidermal appendage (also called primary hair germ; Fig 1.20D). Those cells further differentiate into the inner root sheath (IRS), which later forms the hair shaft, and the outer root sheath (ORS), which is contiguous with the epidermis and surrounded by a BM (Fig 1.20E). Migration of ORS cells along the BM leads to the down growth of the HF into the subcutis until postnatal day 8 (P8) followed by proliferation and differentiation of hair matrix cells into six concentric layers of the IRS and the hair shaft. The development of the HF is

P16 proliferation of the hair matrix cells ceases and the HF degenerates (catagen), rests just below the hair bulge (telogen) until signals from the DP at P24 initiate the formation of a secondary hair germ and the downward migration of ORS cells to form a new HF. This periodic cycling of HFs continues the whole life of the animal (Blanpain and Fuchs 2006).

Fig 1.20. Morphogenesis of the murine HF. A. The HF is formed after a series of dermal-epidermal cues. B.

The condensation of mesenchymal cells induces the formation of an epidermal placode. C. The epidermal placode induces the formation of a DP in the dermis. D. The DP stimulates cell proliferation and differentiation leading to the formation of a hair germ. E. Migration of ORS cells drives the downward growth of the HF, which further differentiates into inner root sheath and hair matrix. The DP remains attached to the HF. (Based on Blanpain and Fuchs 2006).

1.6.3. The role of integrins in the epidermis

The most abundant integrins expressed in basal keratinocytes are α2β1, α3β1, α9β1 and α6β4 integrins. The expression of these integrins is under normal conditions restricted to basal keratinocytes and the outer root sheath cells. While β1 integrins are expressed around the basal keratinocyte, α6β4, a component of hemidesmosomes, is restricted to the basal side adjacent to the BM. In humans, mutations in the genes encoding either α6 or β4 integrin cause an autosomal recessive disorder called epidermis bullosa which is characterized by severe skin blistering. Similarly, α6 or β4 knockout mice die shortly after birth due to epidermal disintegration indicating that the hemidesmosomal α6β4 integrins are essential for cell attachment of basal keratinocytes to the BM (Dowling et al. 1996; van der Neut et al. 1996).

Moreover α6β4 has been implicated in skin carcinogenesis and seems to promote cell migration through mechanisms that involve integrin-RTK crosstalk (Giancotti and Tarone 2003). Deletion of the α3 integrin subunit also results in skin blistering, which is however much less severe as in α6β4-null mice. Interestingly, ablation of both α3 and α6 integrin subunits still allows stratification and HF morphogenesis (DiPersio et al. 2000).

The role of β1 integrin has been addressed by targeted ablation of the protein specifically in basal keratinocytes. The results of these studies will be briefly discussed in the following section.

1.6.3.1. Deletion of β1 integrins from basal keratinocytes

Two groups simultaneously reported the deletion of β1 integrin in basal keratinocytes (Brakebusch et al. 2000; Raghavan et al. 2000). Deletion of β1 integrin leads to skin blistering and complete loss of hair. Animals die within several weeks after birth due to impaired food uptake and disturbed development (Brakebusch et al. 2000) or shortly after birth due to severe skin blistering and dehydration (Raghavan et al. 2000).

Impaired BM maintenance in β1 knockout mice: deletion of β1 integrins in basal keratinocytes resulted in a defective organization of the BM caused by impaired adhesion of basal keratinocytes to the BM and aberrant processing and deposition of ECM proteins such as laminin332 or collagenVII. Although hemidesmosome can form, their number is reduced. The distortion of the dermal-epidermal junction and the reduced adhesion are the reason for the severe skin blistering. The BM of HFs was found to be unaffected, which supports the notion, that skin blistering is boosted by mechanical stress.

Reduced proliferation of basal keratinocytes and hair matrix cells: the proliferation rate of basal keratinocytes is significantly reduced in the absence of β1 although not completely

blocked. Also the proliferation of hair matrix cells essential for the downward growth of the HF is reduced. The survival of basal keratinocytes or cells of the HF is not changed.

Delayed terminal differentiation: β1-deficient epidermis is hyperthickened, caused by a delay in terminal differentiation. Basal keratinocytes, however, did not initiate premature differentiation and maintain their basal properties. This argues against the hypothesis that β1 integrins are essential negative regulators of terminal differentiation.

Altogether these data indicated that β1 integrins are indispensable for the BM integrity along the dermal-epidermal junction. They are essential for the processing and deposition of ECM proteins and necessary for cell attachment of basal keratinocytes. Similarly to the situation in chondrocytes, β1 integrins promote proliferation of keratinocytes but only slightly impair their differentiation.