General Discussion

couple of thousand. Many more gene expression differences have been observed in

instances where there was a greater representation of genes (Fan et al.., 1998). The Unigene

filters had at least 20,000 genes represented on them, •- ZV'-S'P% of the genes in the

genome. However, many of the genes important in epidermal development were not present. Most fundamentally in the analysis of basal kératinocytes was the absence of both |31 integrin and involucrin. The number of potential stem cell markers detected may have been

a

more if skin-specific filter was produced. Efforts in this direction have been made. For example the Dermarray filters by Integriderm (www.integriderm.com). Until the genome is completely sequenced and spotted on one filter or chip, there will always be gaps in the expression data from experiments such as these.

6.4 Further Work

Further in vitro work

There is a requirement for strengthening the data already found in vitro. Where are MRP-

14 and 8 localised in the kératinocytes, is there really nuclear localisation in some cells, and tdnC/iAlM

is the filamentous distribution really with the intermediate filaments? Co-staining with appropriate markers will help to answer these questions, for example co-staining with keratins will prove an intermediate filament distribution. The localisation of E-FABP is not under question, except that there is a need to prove in a robust fashion that some cells have no E-FABP in the nucleus. One potential way of doing this would be by laser scanning cytometry, which has the ability to detect expression of genes in the nucleus of cells by co- staining with a nuclear dye. In this way a better quantification of this phenomenon would be achieved.

The relevance of E-FABP and MCSP as potential markers of transit amplifying cells and stem cell respectively would have to be tested. The clonogenicity of E-FABP over­ expressing cells has to be determined to show that E-FABP may be responsible for driving stem cells into the transit amplifying compartment. There is also a need to demonstrate that MCSP can further enrich for stem cells by proving that the double positive population (pi integrin high, MCSP high), is more clonogenic that the single positive (P1 integrin high,

MCSP low) population. If this were the case, then these two populations would be ideal for further differential expression analyses.

Overexpression of the genes and expression of mutant contructs of these genes examined in this thesis would be the next approach to take. It is unlikely that the overexpression of any of the wild type genes, with the possible exception of MCSP would help in finding out their

function. Both MRP-8 and 14 are calcium binding proteins (Hessian et al, 1993). It would

be interesting to determine the effect of the expression of MRP genes that were insensitive to divalent cations. Do they still produce a filamentous distribution in the suprabasal cells? One problem is that questions concerning hyperplasia are impossible to address in culture as in effect there is a constant state of hyperplasia, for example Keratin 6 is expressed in

cultured kératinocytes (Takahashi et al., 1995).

Overexpression of MCSP in kératinocytes may lead to a higher percentage adopting a stem cell phenotype. However a better way of showing the importance of MCSP in defining keratinocyte stem cells would be by expressing a dominant negative MCSP gene. One potential way would be by replacing the extracellular and transmembrane domains with

CDS, much as has been done with (31 integrin (Zhu et al, 1999). Will the kératinocytes

expressing the dominant negative construct be less clonogenic? How does the expression of (31 integrin change in these cells? Work in melanoma cell lines suggests that there is cross

talk between MCSP and integrins (lida et a l, 1995). Infecting dominant negative pi integrin

and MCSP constructs into the same kératinocytes could begin to address these points.

Differential gene expression approaches

As mentioned earlier, gene expression analyses of BCCs are likely to not yield any more important information on the development of these tumours. However other analysis

techniques, in conjunction with j more useful markers could prove useful in getting more

data on differences in gene expression between stem and transit cells. For example there is potential for MCSP to be used in conjunction with pi integrin to further enrich for stem

cells. Subtraction of cDNA populations or representational difference analysis (Lysitsyn et

Chapter 6 General Discussion

used with a smaller starting population, allowing the possibility of microdissecting the stem and transit cells on the basis of their position in the basal epidermis (stem cells at the dermal papillae, and transit amplifying cells at the rete ridges). Such an approach would eliminate differences that could be due to culture conditions and therefore be more representative of

the situation in vivo.

Differential gene expression analyses will be problematic if the potential differences in expression are of the order of 2-fold. However, the hope is that by enriching the stem cell population as much as possible, the maximum differences in expression levels will be achieved. There is likely to be a limit to the number of differentially expressed genes detectable by differential gene expression approaches. However, over-expression, or expression of dominant negative forms of known candidates by retroviral transduction, combined with a better representation of genes on the filters or possibly microarrays will allow for functional genomics to be performed, i.e. what pathways within basal kératinocytes are responsible for defining the early interfollicular lineages.

Inducible mouse models

In vitro experiments have the disadvantage of being out of context. Inducing or knocking

out expression of genes of choice in the mouse provides an in vivo model of the function of

these genes. Inducible transgenics where the gene of interest is fused to a mutant oestrogen

receptor activatable by tamoxifen (Littlewood et ai, 1995) and tissue specific knockouts,

using Cre-Lox technology (Reviewed Sauer 1998), are commonplace. Even more recently a

combination of the two has been attempted (Vasioukhin et al., 1999), using an inducible

Cre. One example of the inducible gene approach is the activation of c-Myc by tamoxifen

in the basal epidermis driving stem cells to become transit amplifying cells, with the cells differentiating to become sebocytes and interfollicular epidermis (Arnold and Watt, submitted)

MRP-8 and 14 are expressed in all instances of hyperproliferation. Is it a cause or an effect? Does MRP-8 and 14 induce hyperplasia? It seems likely that an inducible transgenic approach would be best suited to answer this question. Conversely knocking out MRP-8 or

14 in the suprabasal epidermis and then subjecting the skin of the mouse to wounding or neoplasia could explain the reason for the expression of these genes under those circumstances. E-FABP is ubquitously expressed and would have to be knocked out in the basal layer of the epidermis only, to maintain its potentially different function in the granular layer. It would be interesting with these mice to test the hypothesis that E-FABP drives cells into the transit compartment, with the expectation that there would be fewer transit cells in the epidermis. As E-FABP is a downstream target of c-Myc, mobilisation of stem cells could be altered as a result. It would also be of interest to examine the expression of E- FABP in the inducible c-Myc transgenic of Arnold and Watt. The expectation would be that expression would increase in treated epidermis - consistent with the results of Coller et al (2000) in fibroblasts.

The NG2 null mouse (Grako et a l, 1999; Burg et a/., 1999) has no obvious skin phenotype,

contrasting with the mice in which pi integrin is absent in the basal epidermis, which have

severe effects on both the skin and the hair follicles (Brakebusch et al, 2000, Raghavan et

al., 2000). Grafting transgenic human epidermis onto nuje mice maybe one way of approaching the function of MCSP in the epidermis, by comparing the aforementioned dominant negative kératinocytes to kératinocytes expressing wild-type MCSP. A further possibility would be to cross the pi integrin epidermal knockout mice with the NG2 null mouse, or a basal layer specific MCSP knockout mouse. This would provide insight into the possiblity of MCSP and pi integrin cooperating in some way to define the stem cell compartment.

6.5 The Future

There are many factors that determine the fate of basal kératinocytes during normal development and tumorigenesis. Determining the genes that are expressed in stem cells and transit cells, is just one step on the path of defining completely the phenotype and expressed genome of these cells. Stem cells signal to surrounding cells, one such example being the

notch pathway (Lowell et al, 2000). These signals between adjacent cells, and other

transient signals that determine stem cell fate, ^[^potentially are altered during

Chapter 6 General Discussion

populations. In addition there are potential cues from the dermis and basement membrane that maintain stem cells. Potential examples could be members of the Hedgehog pathway and the human homologue of the wingless pathway, which signals through (3-catenin. Finally, in the context of the stem cell and transit amplifying cells, the relevance of genes with interesting expression patterns in the dermis and basement membrane will have to be

determined. For example laminin y3 is expressed in the regions overlying the dermal

papillae (Koch et al, 1999). The keratinocyte stem cell, like all stem cells, is a product of its

own gene expression and the interactions with its neighbours and microenvironment. The technologies are now in place to help address all of these factors and their interactions. The hope is that understanding of the control of stem cell fate in the epidermis wiU provide insight not only into the function the epidermis, but the whole range of epidermal neoplasias.