EUROPEAN TISSUE REPAIR SOCIETY

STEM CELLS I

SKIN DIFFERENTIATION DURING EMBRYOGENESIS: STEM CELLS AND DERMAL-EPIDERMAL INTERACTIONS

THE skin is an excellent model system for studying the control and determination of cell fate. Two distinct cell compartments, the dermis and epidermis, of different embryonic origin, interact to give rise to the interfollicular epidermis and the follicular appendages. Whereas the epidermis arises chiefly from the ectoderm, the dermis is derived from several sources, including the neural crest, paraxial mesoderm and lateral mesoderm. Thus, for example, in the chick the mediodorsal dermis is formed by Wnt-11 expressing cells that migrate from the mediodorsal lip of the dorsal part of the somite (the dermomyotome) under the control of a Wnt-1 signal originating from the neural.^1,2 The migration of dermal progenitors under the ectoderm, which transforms into an epidermis, gives rise to the embryonic skin.

Once in place, the two cell populations engage in a complex exchange of signals, including the hedgehog, wnt/ β-catenin and notch/delta systems, which serve to further organise the tissue and specify the fate of the two cell populations.^3–9 This includes the formation of epidermal appendages, such as hair in mammals, or feathers and scales in birds. The appendage-forming properties of a particular region of dermis are pre-specified in the presomitic or somatopleural mesoderm before the migration of dermal progenitors.¹⁰ The expression of this developmental programme, however, requires a permissive signal from the epidermis, probably a member of the FGF-family.³

In both mammalian and avian species, appendages appear initially as epidermal placodes and dermal condensations that then give rise to the hair or feather bud, which then differentiates into a mature appendage. The distribution of members of the Notch/Delta signalling pathway during hair morphogenesis suggests that this system has a role in determining the segregation of the future stem cell compartment.¹¹ Even after embryonic development is complete, these structures undergo a cycle of growth and renewal, which recapitulates to a large degree their initial development. Hairs have been shown to contain reservoirs of multipotent stem cells that are important for normal homeostasis and for wound healing in cases of injury.^12,13

In our laboratory therefore, we have used the skin and related structures of chick and mammals as a model system to study aspects of cell determination and fate choice. In particular, what are the mechanisms of the dermal and epidermal fate determination and, how and when are the stem cells in these tissues specified? It has been known for some time that embryonic dermis is able to induce appendage formation,¹⁴ but our recent results imply that this process also involves the induction and segregation of novel populations of stem cells associated with these new appendages.

Epidermal cell plasticity

It has been established that epidermal cells, regardless of their origin, are capable of responding to the inductive signals of the embryonic dermis,¹⁴ or even the dermal component of the adult hair follicle (the dermal papilla),¹⁵ by giving rise to new hair. Which are the cells in the adult epidermis that respond to the dermal signals to give rise to the new structures has been, however, unclear. Are these resident, multipotent stem cells or can cells that have begun to differentiate, for example transient amplifying (TA) cells, also respond to these signals by reverting to an undifferentiated state and changing their developmental programme? This would imply a greater degree of plasticity in these cells than has hitherto been recognised. In the interfollicular epidermis this is difficult to determine as there appear to be secondary stem cells dispersed and intermingled with the transient cells throughout the basal layer of the epidermis^5,8,16 (Figure 1B). The primary source of multipotent stem cells in the epidermis rests in the bulge region of the hair follicle.^12,17 In the eye, the stem cell reservoir which gives rise to the mature central cornea epithelium in the adult appears to be segregated into a distinct region on the periphery of the cornea called the limbus^18,19 (Figure 1A). There are apparently no secondary stem cells in the central cornea as the cells in this region express differentiation specific markers such as keratins 3 and 12. This segregation of stem and TA cells allows us to perform experiments involving the recombination of embryonic dermis at the stage of dermal condensation (from the mouse) with rabbit central cornea epidermis that is devoid of stem cells.²⁰ The results show that the embryonic dermis induces the formation of new hair follicles and interfollicular epidermis from the differentiated cells from the central cornea. A closer examination of the kinetics confirms and highlights the mechanism by which this process occurs.

Figure 1. Localisation of stem cells in corneal epithelium and epidermis.

(A) The corneal epidermis has a primary stem cell reservoir located in the basal layer of the limbus and transient cells dispersed throughout the basal layers of the central cornea.
(B) The epidermis has a primary stem cell reservoir in the bulge region of the hair follicle and secondary stem cells located in the matrix region of the hair follicle and distributed throughout the basal layer of the interfollicular epidermis.

The initial recombined central corneal epithelium consists solely of differentiated late transient amplifying cells which show limited proliferative capacity in culture¹⁹ and express the differentiation specific keratins K12 and K3 and not K5 and K14 (Figure 2A). After recombination and grafting, the epithelial tissue undergoes significant rearrangement. Initially the tissue is disorganised and uniformly positive for K12 (Figure 2B), but by four days a new basal layer is formed of cells that down-regulate K12 (Figure 2C). At later stages this new basal layer begins to express the basal keratin K5 (Figure 2D), similarly to the basal layer of the skin and the limbus. Subsequently hair pegs are formed. The reorganization of the basal layer is coupled with an increase in the expression and localization of β1-integrin at the basal plasma membrane (data not shown).

Figure 2. Expression of keratin 5 and keratin 12 at the limbus/central cornea junction in normal rabbit eye (A), and corneal epithelium/ dermal recombinants (C–D).

K5 was detected using a monoclonal primary (AE14) and an alexa 488 labeled anti-mouse secondary antibody (green). Subsequently K12 was detected with a rhodamine-conjugated monoclonal antibody (AK12) (red). Nuclei were counterstained with Hoechst 33258 (blue).
(A) The expression of K5 and K12 in the adult rabbit eye. Note the loss of K5 expression and uniform expression of K12 in the central cornea as compared to the limbus.
(B) Two days after recombination of rabbit central corneal epithelium and mouse embryonic dermis. Note the disorganisation of the epithelium and the uniform expression of K12.
(C) Four days after recombination a K12-negative basal layer is formed.
(D) By day nine there is a well-organised K12-negative basal layer that strongly expresses K5. Dashed lines indicate the mesenchymal/epithelial junction, dotted lines the junction between the limbus and central cornea.

After two weeks, mature hair follicles, including the hair shaft and associated structures such as the dermal papillae and sebaceous glands, are formed. In addition, K10-expressing cells appear, initially at the hair/epithelium junction (Figure 3), and finally a fully differentiated interfollicular epidermis, including the granular and cornified layers, is formed. The appearance of the new K12-/K5+ basal layer is apparently not a consequence of the division of K12+ cells but represents a down-regulation of the K3/12 pair and subsequent up regulation of the K5/14 pair in these cells which can then participate in the formation of hair and interfollicular epidermis. This would thus represent another example of transdifferentiation.21 These dedifferentiated cells are then able to undergo cell division and participate in the formation of new structures, including hair and interfollicular epidermis, which are defining characteristics of stem cells.

Figure 3. Formation of hair follicles and appearance of keratin 10 in corneal epithelium/dermal recombinants. K10 was detected using a rat monoclonal primary antibody and an alexa 488 labeled anti-rat secondary antibody (green). Subsequently K12 was detected with a rhodamineconjugated monoclonal antibody (AK12) (red). Nuclei were counterstained with Hoechst 33258 (blue). Note the appearance of the K10 expressing cells at the junction of the hair follicles and the epithelium and the lack of K12 staining in the basal layer and hair follicles.

Conclusions

In summary, studying the steps of skin and skin appendage development, in particular the complex interaction and cross talk between the dermis and the epidermis can give important insight into the processes involved in normal and abnormal skin homeostasis, including wound healing. The recognition that the dermis is derived from multiple sources and hence is not homogenous throughout the body is an important factor to take into account when designing skin replacements and grafts. In particular, the fact that there is an absolute requirement for epidermal signalling to allow the formation and maintenance of dermal inductive capabilities is vital to the development of grafting and artificial skin techniques that could serve as true skin replacements complete with appropriate epidermal appendages.

Our work on transdifferentiation of transient amplifying cells supports the idea of a hierarchy of ‘stemness’ in epidermal cells as suggested by Christopher Potten.22 This model suggests that, as well as there being a reservoir of stem cells in the skin, differentiating transient cells are capable of undergoing dedifferentiation to regain stem cell characteristics. This implies that the degree of plasticity, and hence the capability to adopt new cell fates or even revert to stem cells, is a function not only of intrinsic factors within the cell but also of the external environment. Thus cells that have left the stem cell niche, and are thus apparently committed to differentiation, i.e. transient amplifying cells, are capable or responding to changing circumstances and inductive signals by reversing their differentiation and adopting new fates (Figure 4). The identification of significant plasticity in epidermal cells thus opens up exciting new avenues of investigation for the treatment of acute and chronic skin injuries and perhaps even for cell or in situ cytokine therapy.

Figure 4. Model of stem cell formation and maintenance. Pluripotent embryonic stem cells in the early blastocyst undergo several differentiation events during the course of development. The first of these is to form the cells of the three embryonic layers that further differentiate into tissues. Within each tissue, particularly those that undergo regular turnover such as skin or intestinal epithelium, stem cell compartments are formed that are maintained in the adult organism. These will give rise to transient amplifying cells that undergo differentiation to give rise to the mature tissue and serve as a reservoir for tissue homeostasis and repair. At each of these steps, the differentiation potential of the stem cells is restricted. Recent experiments have demonstrated, however, that adult tissue stem cells, or even some differentiating cells, when exposed to a permissive or instructive environment, can reverse their phenotype to become stem cells with a less restricted differentiation potential.

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Corresponding authors:
Pearton, D. J. and Dhouailly, D.
Biologie de la Différentiation Epithéliale,
UMR CNRS 5538,
Institut Albert Bonniot,
Grenoble, France