Degenerate primer PCR was carried out by Phil Jones in the lab to identify Delta homologues expressed by cultured human kératinocytes, Six out of six clones
sequenced corresponded to human Deltal (Gray et a l, 1999) (Figure 4.1). Neither of the two other members of the vertebrate Delta family, D113 or D114, were detected.
In situ hybridisation of sections of human foetal and adult skin, carried out by the ICRF in situ hybridisation service, showed that Deltal mRNA was confined to the basal layer of the epidermis and absent from the underlying dermis (Figure 4.2 A-D). Expression within the basal layer was not uniform; rather, there were groups of cells that had higher levels of mRNA than their neighbours and these groups tended to lie at the tips of the dermal papillae, where the underlying dermis comes closest to the surface of the skin. In situ hybridisations for Notchl were not performed because good monoclonal
antibodies were already available: The Deltal in situ hybridisations were carried out at a time when the Deltal antibody had not yet been made.
Immunofluorescence labelling of neonatal and adult epidermis with a polyclonal antibody to chick Deltal also showed expression confined to the basal layer. The staining intensity varied within the basal layer in the same was as found by in situ hybridisation (Figure 4.2E)
In contrast to Deltal, Notchl protein was detected in all the living layers of the neonatal and adult epidermis, the staining being weak in the basal layer and most intense in the suprabasal layers (Figure 4.2F). Two different monoclonal antibodies against Notchl (Tan20 and Tan 18) gave identical staining patterns. Notch 2 was barely detectable in human epidermis by immunostaining (not shown). Notch 3 and 4 were not examined.
This expression pattern is consistent with Northern blots of Notchl and Deltal
expression in cultured human epidermal kératinocytes, carried out by Phil Jones (Figure 4.3). Populations were enriched for stem cells (S), transit amplifying cells (TA) or suprabasal, terminally differentiating cells (TD) by differential adhesiveness to type IV collagen as described in section 2.2.6. Blots were probed for involucrin, a marker of terminal differentiation that is selectively expressed in suprabasal kératinocytes (Jones and Watt, 1993) and, as a loading control, for IBS RNA. Two transcripts
C H A P T E R 4___________S t e m C e l l D i f f e r e n t i a t i o n ____________
(approximately 4.0 and 4.6 kb) were detected with a probe to human Deltal, potentially reflecting differences in polyadenylation (Gray et a l, 1999). Deltal mRNA was
detected in stem and transit amplifying cells, but was virtually undetectable in
terminally differentiating cells. When the Deltal signal was quantitated relative to the 18S RNA loading control, the mRNA was approximately two fold more abundant in stem cells than transit amplifying cells (1.7 fold in Figure 4.3 A; average of two fold in three independent batches of kératinocytes), consistent with the in situ hybridisation results in Figure 4.2 A-D. Notchl mRNA (transcript size approximately lOkb) was detectable in stem cells, but was upregulated in terminally differentiated kératinocytes (Figure 4.3B),
4.2.2, Retroviral transduction o f kératinocytes
There are two widely used approaches to manipulating Notch function. One is to missexpress constitutively active or dominant negative forms of Notch (Rebay et a l,
1993), another is to missexpress normal or dominant negative forms of the ligands that activate Notch (Henrique et a l, 1997). I took the second approach, with the view that activation of Notch by the ligand might be more physiologically relevant than using constitutively active Notch mutants, and that Notch might respond differently to different ligands.
Construction o f retroviral vectors
cDNAs were obtained from Isabelle Le Roux (Developmental Genetics Lab ICRF) and Jenny Dunne (Lymphocyte Activation Lab, ICRF) that encode full length mouse Deltal (bp 14 to 2190) and a truncated mutant, DeltaT, lacking all but 13 of the amino acids in the intracellular domain (bp 14 to 1756). This mutants act as a dominant negative inhibitor of Notch activation, rendering cells unable to respond to Delta signals from their neighbours (Henrique et a l, 1997). Mouse Deltal shows close homology to human Deltal (Figure 4.1)
A DNA binding mutant of Xenopus Suppressor of Hairless, SuH^®”^, was obtained from Chris Kintner (The Salk Institute, La Jolla). This mutant acts as a dominant negative inhibitor of the Notch signalling pathway since it binds to activated Notch but is unable to bind DNA and so cannot activate transcription of target genes (Wettstein et a l,
1997). This mutant was modified by Isabelle Le Roux (ICRF) using PCR to include an HA tag at the 3’ end.
I subcloned these cDNAs into a high titre, direct orientation retroviral vector, pBabe puro (see section 2.5.5.). For the Deltal vector I constructed both untagged and IRES-
C H A P T E R 4__________ S t e m C e l l D i f f e r e n t i a t i o n ____________
EGFP tagged versions. The 1RES encodes an internal ribosomal entry site: the Delta 1- IRES-EGFP is therefore transcribed as one continuous strand of mRNA but is translated as two separate proteins (Pear et a l, 1998). As a control I constructed a vector
containing only the 1RES-EGFP.
The IRES-EGFP tagged version of the construct was used for infection of 3T3-J2 cells. The tag was needed because 3T3-J2 cells tended to lose expression of Deltal over time, even though they did not lose puromycin resistance. Cells that lose Deltal expression will also lose EGFP expression, and so the tag allows the Deltal-negative population to be eliminated by FACS sorting on the basis of EGFP fluorescence. It is not clear why J2 fibroblasts tend to lose expression of Deltal, but this seems to be a problem specific to the Deltal protein since other proteins, for example EGFP, can be stably expressed by J2 cells without any need of further selection. Kératinocytes maintained good expression of Deltal over several passages, as monitored by immunostaining, without the need for further selection, and so the EGFP tag was not necessary and untagged constructs were used in all keratinocyte infections.
Construction o f retroviral producer cells
Both Ecotropic retroviral producer cell lines, GPE, and amphotropic producer lines, AM 12, were generated (see section 2.1.5). Although clonal cell lines were made for the Delta AM 12 producer cells, none of the 24 screened by immunostaining had expression levels that were as high as the polyclonal population, and so polyclonal lines were used for all infections (Figure 4.4 A-B). For the IRES-EGFP tagged ecotropic producer cells, the population with the top 20% EGFP expression was selected by FACS.
Immunostaining with the anti-Deltal antibody confirmed that every EGFP positive cell was positive for Deltal (Figure 4.4 D). This virus was only used for infecting J2-3T3 cells, which are mouse cells, and so it was not necessary to make amphotropic virus producer cells. Clones of SuH°®^ AM 12 virus producer cells were screened by immunostaining for the HA tag. One of two selected clones is shown in Figure 4.4 F. Only early passages of retroviral producer cells were used for infection of primary human kératinocytes and J2-3T3 cells.
Transduction o f human epidermal kératinocytes with Delta retroviruses
The standard protocol used to transduce primary human epidermal kératinocytes with retrovirus is to co-cultivate the kératinocytes with amphotropic producer cells which have been treated with mitomycin C (Rheinwald and Green, 1975). I found that Delta producer cells tended to promote differentiation of co-cultivated kératinocytes, making it very difficult to obtain actively growing infected populations. A likely explanation for this, in the light of the findings described later in this chapter is that Deltal protein
C H A P T E R 4__________ S t e m C e l l D i f f e r e n t i a t i o n ____________
on the surface of the producer cells activates the Notch pathway in adjacent kératinocytes. I overcame this problem by using retroviral supernatant to infect kératinocytes. The proportion of cells transduced in this way was close to 100%, both on first infection and in subsequent passages. Immunofluorescence was used to compare the level and subcellular localisation of transduced and endogenous Deltal (Figure 4.5)
When overexpressed, Deltal and DeltaT accumulated at cell-cell borders (Figure 4.5 A,B). The antibody that we used was raised against a peptide region of Deltal that is highly conserved between mouse and human (this region is highlighted on the Deltal sequence alignment in Figure 4.1) and could also therefore be used to detect the
endogenous protein (Figure 4. IE). In empty vector infected kératinocytes, endogenous Deltal was weakly expressed and was localised in a punctate distribution at cell-cell borders (Figure 4.5 C). The overexpressed proteins therefore accumulated at the same cellular location as the endogenous protein.
4.2.3. Effects o f uniformly overexpressed D eltal or DeltaT on keratinocyte growth