Poblaci´ on inicial

In contrast to the severe HF phenotype, the epidermis of rac1fl/fl,cre mice seemed to be not affected by the loss of Rac1 (3.1.3 and Fig. 3.9-Fig. 3.12). The histological analysis (2.7.1) of the 14-day-old mutant skin did not reveal evident defects, such as an increased amount of layers or detachment of the Rac1-deficient epidermis form the dermis (Fig. 3.25, HE).

Fig. 3.24 Normal morphology and proliferation of the Rac1-deficient epidermis. HE staining of back skin sections from 14-day-old control and mutant mice showed no defects of the epidermis in the absence of Rac1. No changes in the proliferation of basal keratinocytes as well as no abnormal proliferation of suprabasal cells were detected with a BrdU-incorporation assay in the mutant epidermis. Bars: 25 µm.

Mitotic activity of epidermal cells in the 14-day-old skin was analysed using a BrdU incorporation assay (2.7.2.2). The proliferation rate of basal keratinocytes in the mutant epidermis was similar to the control value (ctrl: 10.3% BrdU+ cells; KO: 11.4% BrdU+ cells). Also, no proliferation of Rac1-deficient suprabasal cells was detected (Fig. 3.24, BrdU). Those results were in agreement with the normal morphology of the Rac1-deficient epidermis.

In some older mice, regions with a slight thickening of the epidermis were observed, suggesting a local increase in the proliferation.

3.3.1

Normal differentiation of the epidermis

A normal layer composition of the epidermis in the absence of Rac1 was confirmed by the IF expression analysis of differentiation-specific keratins (2.7.2.1). Distribution patterns of K14 and K10, which are typical for basal and suprabasal keratinocytes, respectively (1.2.1), were comparable in the 14-day-old control and mutant epidermis (Fig. 3.25, K14 and K10). Also the presence of loricrin, which is normally expressed in terminally differentiated keratinocytes, was restricted to the cornified layer of the epidermis as in control samples (Fig. 3.25, loricrin).

The expression of K6 is induced in the epidermis only when the normal biology of keratinocytes is disturbed, e.g. under hyper- or hypoproliferative conditions (Dominey et al.,

1993; Sellheyer et al., 1993). However, as in control samples, no K6 expression was detected in the mutant epidermis (Fig. 3.25, K6), further indicating that the absence of Rac1 does not lead to pathological changes.

Fig. 3.25 Normal differentiation of the epidermis in the absence of Rac1. IF staining of back skin sections from 14-day-old control and mutant mice were carried out for K14, K10, loricrin and K6 to analyse differentiation of the epidermis. A counterstaining for α6 integrin was performed to visualize the dermal-epidermal junction. Nuclei were visualized with DAPI. Bars: 25 µm.

3.3.2

Normal expression of adhesion and ECM proteins

In vitro studies with primary keratinocytes showed that Rac1 is required for the establishment and maintenance of the cadherin-mediated cell–cell adhesion (Braga et al., 1997; Braga et al., 1999). Surprisingly, IF staining of junctional proteins showed normal expression patterns and the intracellular distribution of E-cadherin, β-catenin, α-catenin and desmoplakin in the 14-day-old mutant epidermis (Fig. 3.26) giving no indication of a defect in the cell-cell adhesion between Rac1-deficient keratinocytes in vivo.

Furthermore, Rac1 was shown to be necessary for the assembly of laminin at the basal side and for the establishment of cell polarity in MDCK epithelial cysts grown in collagen (O'Brien et al., 2001). However, the deposition of BM components such as LN5 and nidogen (Nd) was not altered in the 14-day-old mutant skin and remained restricted to the dermal-epidermal junction (Fig. 3.27, LN5 and Nd). Like in the control skin, the BM was linear and continuous.

adherent to the BM, in both control and mutant epidermis, demonstrating that basal keratinocytes were polarized normally in the absence of Rac1 (Fig. 3.27, β4).

Fig. 3.26 Normal expression of cell-cell contact components in the Rac1-deficient epidermis. IF staining of back skin sections from 14-day-old control and mutant mice was carried out for E-cadherin

(E-cad), β-catenin (β-cat), α-catenin (α-cat) and desmoplakin (desm) to analyse the expression of

adhesion proteins in the epidermis. A counterstaining for α6 integrin was performed to visualize the dermal-epidermal junction. Bars: 25 µm.

Fig. 3.27 Normal deposition of the BM and polarization of basal keratinocytes in the absence of Rac1. IF staining of back skin sections from 14-day-old control and mutant mice was carried out for LN5 and Nd to analyse the deposition of BM components. Polarization of basal keratinocytes was shown by visualizing the expression pattern of integrin α6β4. Bars: 25µm.

3.3.3

Normal formation of cell-cell and cell-ECM contacts

An ultrastructural analysis of back skin (2.7.3) was performed to study in more detail the structure of cell-cell contacts and the attachment of basal keratinocytes to the BM.

EM pictures showed a normal presence of desmosomes (Fig. 3.28A and B, black arrows) between Rac1-deficient basal keratinocytes (K), which were accompanied by adherens junctions, as in the control epidermis (Fig. 3.28B, white arrowheads). Those results further indicated that the cell-cell adhesion between basal keratinocytes in vivo is not affected by the absence of Rac1.

Fig. 3.28 No apparent defects of cell-cell and cell-ECM contacts in the Rac1-deficient epidermis.

(A) EM pictures of back skin sections from 14-day-old control and mutant mice confirmed a normal

presence of desmosomes (black arrows) between Rac1-deficient basal keratinocytes (K) attached to

the BM (black arrowheads). Bar: 1 µm. Higher magnification EM pictures showed: (B) normal

adherens junctions (white arrowheads) formed next to desmosomes (black arrows) and (C) normal

hemidesmosomes (white arrows) formed between basal keratinocytes and the BM (black arrowheads) in the absence of Rac1. Bars: 0.5 µm.

when compared to control samples. However, that seemed to have no influence on the maintenance of adhesion structures.

The EM analysis confirmed a normal deposition of the BM in the absence of Rac1 (Fig. 3.28A and C, black arrowheads). In addition, higher magnification EM pictures showed a presence of hemidesmosomes in both control and mutant skin (Fig. 3.28C; white arrows). No detachment of the epidermis from the underlying BM was observed in the Rac1-deficient skin, as expected from the histological analysis.

Those data showed that adhesion capability of mutant keratinocytes in vivo was either not affected at all by the absence of Rac1 or, at the least, that it was not altered enough to cause a noticeable defect.