Catherin Niemann

Manipulation of stem cell proliferation and lineage commitment:visualisation of label-retaining cells in wholemounts of mouse epidermis

Mammalian epidermis is maintained by stem cells that have the ability to self-renew and generate daughter cells that differentiate along the lineages of the hair follicles, interfollicular epidermis and sebaceous gland. As stem cells divide infrequently in adult mouse epidermis, they can be visualised as DNA label-retaining cells (LRC). With whole-mount labelling, we can examine large areas of interfollicular epidermis and many hair follicles simultaneously, enabling us to evaluate stem cell markers and examine the effects of different stimuli on the LRC population. LRC are not confined to the hair follicle, but also lie in sebaceous glands and interfollicular epidermis. LRC reside throughout the permanent region of the hair follicle, where they express keratin 15 and lie in a region of high α6β4 integrin expression. LRC are not significantly depleted by successive hair growth cycles. They can, nevertheless, be stimulated to divide by treatment with phorbol ester, resulting in near complete loss of LRC within 12 days. Activation of Myc stimulates epidermal proliferation without depleting LRC and induces differentiation of sebocytes within the interfollicular epidermis. Expression of N-terminally truncated Lef1 to block β-catenin signalling induces transdifferentiation of hair follicles into interfollicular epidermis and sebocytes and causes loss of LRC primarily through proliferation. We conclude that LRC are more sensitive to some proliferative stimuli than others and that changes in lineage can occur with or without recruitment of LRC into cycle.

Rac1 Is Crucial for Hair Follicle Integrity but Is Not Essential for Maintenance of the Epidermis

ABSTRACT Rac1 is a small GTPase that regulates the actin cytoskeleton but also other cellular processes. To investigate the function of Rac1 in skin, we generated mice with a keratinocyte-restricted deletion of the rac1 gene. Rac1-deficient mice lost nearly all of their hair within a few weeks after birth. The nonpermanent part of mutant hair follicles developed constrictions; lost expression of hair follicle-specific keratins, E-cadherin, and α6 integrin; and was eventually removed by macrophages. The permanent part of hair follicles and the sebaceous glands were maintained, but no regrowth of full-length hair follicles was observed. In the skin of mutant mice, epidermal keratinocytes showed normal differentiation, proliferation, cell-cell contacts, and basement membrane deposition, demonstrating no obvious defects of Rac1-deficient epidermis in vivo. In vitro, Rac1-null keratinocytes displayed a strong spreading defect and slightly impaired adhesion. These data show that Rac1 plays an important role in sustaining the integrity of the lower part of hair follicles but not in maintenance of the epidermis.

Human skin stem cells and the ageing process

In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. Despite accumulation of senescence markers in aged skin, epidermal stem cells are maintained at normal levels throughout life. Therefore, skin ageing is induced by impaired stem cell mobilisation or reduced number of stem cells able to respond to proliferative signals. In the skin, existence of several distinct stem cell populations has been reported. Genetic labelling studies detected multipotent stem cells of the hair follicle bulge to support regeneration of hair follicles but not been responsible for maintaining interfollicular epidermis, which exhibits a distinct stem cell population. Hair follicle epithelial stem cells have at least a dual function: hair follicle remodelling in daily life and epidermal regeneration whenever skin integrity is severely compromised, e.g. after burns. Bulge cells, the first adult stem cells of the hair follicle been identified, are capable of forming hair follicles, interfollicular epidermis and sebaceous glands. In addition, -- at least in murine hair follicles -- they can also give rise to non-epithelial cells, indicating a lineage-independent pluripotent character. Multipotent cells (skin-derived precursor cells) are present in human dermis; dermal stem cells represent 0.3% among human dermal foreskin fibroblasts. A resident pool of progenitor cells exists within the sebaceous gland, which is able to differentiate into both sebocytes and interfollicular epidermis. The self-renewal and multi-lineage differentiation of skin stem cells make these cells attractive for ageing process studies but also for regenerative medicine, tissue repair, gene therapy and cell-based therapy with autologous adult stem cells not only in dermatology. In addition, they provide in vitro models to study epidermal lineage selection and its role in the ageing process.

Impaired epidermal wound healing in vivo upon inhibition or deletion of Rac1

To address the functions of Rac1 in keratinocytes of the basal epidermal layer and in the outer root sheath of hair follicles, we generated transgenic mice expressing a dominant inhibitory mutant of Rac, N17Rac1, under the control of the keratin 14 promoter. These mice do not exhibit an overt skin phenotype but show protracted skin wound re-epithelialization. Investigation into the underlying mechanisms revealed that in vivo both proliferation of wound-edge keratinocytes and centripetal migration of the neo-epidermis were impaired. Similar results were obtained in mice with an epidermis-specific deletion of Rac1. Primary epidermal keratinocytes that expressed the N17Rac1 transgene were less proliferative than control cells and showed reduced ERK1/2 phosphorylation upon growth factor stimulation. Adhesion, spreading, random migration and closure of scratch wounds in vitro were significantly inhibited on collagen I and, to a lesser extent, on fibronectin. Stroboscopic analysis of cell dynamics (SACED) of N17Rac1 transgenic and control keratinocytes identified decreased lamella-protrusion persistence in connection with increased ruffle frequency as a probable mechanism for the observed impairment of keratinocyte adhesion and migration. We conclude that Rac1 is functionally required for normal epidermal wound healing and, in this context, exerts a dual function – namely the regulation of keratinocyte proliferation and migration.

TCF/Lef1 activity controls establishment of diverse stem and progenitor cell compartments in mouse epidermis

Mammalian epidermis consists of the interfollicular epidermis, hair follicles (HFs) and associated sebaceous glands (SGs). It is constantly renewed by stem and progenitor cell populations that have been identified and each compartment features a distinct mechanism of cellular turnover during renewal. The functional relationship between the diverse stem cell (SC) pools is not known and molecular signals regulating the establishment and maintenance of SC compartments are not well understood. Here, we performed lineage tracing experiments to demonstrate that progeny of HF bulge SCs transit through other SC compartments, suggesting a hierarchy of competent multipotent keratinocytes contributing to tissue renewal. The bulge was identified as a bipotent SC compartment that drives both cyclic regeneration of HFs and continuous renewal of SGs. Our data demonstrate that aberrant signalling by TCF/Lef1, transcription factors crucial for bulge SC activation and hair differentiation, results in development of ectopic SGs originating from bulge cells. This process of de novo SG formation is accompanied by the establishment of new progenitor niches. Detailed molecular analysis suggests the recapitulation of steps of tissue morphogenesis.

Dual role of inactivating Lef1 mutations in epidermis: tumor promotion and specification of tumor type.

The NH(2) terminus of LEF1 is frequently mutated in human sebaceous tumors. To investigate how this contributes to cancer, we did two-stage chemical carcinogenesis on K14DeltaNLef1 transgenic mice, which express NH(2)-terminally truncated Lef1 in the epidermal basal layer. Transgenic mice developed more tumors, more rapidly than littermate controls, even without exposure to tumor promoter. They developed sebaceous tumors, whereas controls developed squamous cell carcinomas. K14DeltaNLef1 epidermis failed to up-regulate p53 and p21 proteins during tumorigenesis or in response to UV irradiation, and this correlated with impaired p14ARF induction. We propose that LEF1 NH(2)-terminal mutations play a dual role in skin cancer, specifying tumor type by inhibiting Wnt signaling and acting as a tumor promoter by preventing induction of p53.

Alternative Proteolytic Processing of Hepatocyte Growth Factor during Wound Repair

Wound healing is a crucial regenerative process in all organisms. We examined expression, integrity, and function of the proteins in the hepatocyte growth factor (HGF)/c-Met signaling pathway in normally healing and non-healing human skin wounds. Whereas in normally healing wounds phosphorylation of c-Met was most prominent in keratinocytes and dermal cells, in non-healing wounds phosphorylation of c-Met was barely detectable, suggesting reduced c-Met activation. In wound exudates obtained from non-healing, but not from healing wounds, HGF protein was a target of substantial proteolytic processing that was different from the classical activation by known serine proteases. Western blot analysis and protease inhibitor studies revealed that HGF is a target of neutrophil elastase and plasma kallikrein during skin repair. Proteolytic processing of HGF by each of these proteases significantly attenuated keratinocyte proliferation, wound closure capacity in vitro, and c-Met signal transduction. Our findings reveal a novel pathway of HGF processing during skin repair. Conditions in which proteases are imbalanced and tend toward increased proteolytic activity, as in chronic non-healing wounds, might therefore compromise HGF activity due to the inactivation of the HGF protein and/or the generation of HGF fragments that ultimately mediate a dominant negative effect and limit c-Met activation.

Development and homeostasis of the sebaceous gland.

The important role of epidermal appendages especially the sebaceous gland has only recently been recognized. In particular, it has been convincingly shown that normal development and maintenance of the sebaceous gland are required for skin homeostasis since atrophic sebaceous glands and disturbances in sebaceous lipid composition result in major defects of the physiological barrier and maintenance of the skin. Consequently, it is important to unravel the signaling network controlling proper sebaceous lineage differentiation in mammalian skin and to understand the underlying mechanisms leading to severe skin diseases, including abnormal proliferation and differentiation of the gland, defects of the lipid metabolism and barrier, as well as sebaceous tumor formation. Over the last years, results from transgenic and knock out mouse models manipulating distinct signaling pathways in the skin as well as the detailed analysis of human sebaceous gland-derived cell lines provided new insights into crucial mediators balancing proliferation and differentiation of the sebaceous gland. Here, we discuss our current knowledge of in vivo mechanisms of sebaceous gland development, maintenance and disorders and highlight recent contributions to the field of sebaceous gland biology.

Stem cell dynamics in sebaceous gland morphogenesis in mouse skin.

The hair follicle (HF) and the sebaceous gland (SG) constitute the two integral parts of the pilosebaceous unit and significantly contribute to the barrier function of mammalian skin. Considerable progress has been made in our understanding how HF formation is regulated. However, the development of the SG is poorly understood, both at the molecular and cellular level. Here, we investigate the process of SG morphogenesis and the dynamics of its cellular organisation in more detail. The spatial and temporal organisation of distinct stem and progenitor compartments was analysed during morphogenesis of the pilosebaceous unit in mouse tail epidermis. Our experiments reveal a dynamic expression pattern for diverse HF stem cell marker molecules including Sox9 and Lrig1. Surprisingly, Sox9 and Lrig1 are initially coexpressed by epidermal progenitor cells and are confined to different regions within the pilosebaceous unit when the specification of the sebocyte cell lineage takes place. We demonstrate that SG development at the distal part of the HF is driven by asymmetric cell fate decision of Lrig1 positive stem cells, whereas MTS24/Plet1 positive precursor cells seem not to play a role in this process. Importantly, our data clearly show that distinct stem and progenitor compartments are established at different time points of development. By studying the process of SG morphogenesis more precisely, we discovered that the two prominent SGs attached to one tail HF originate from one small cluster of sebocyte cells. Finally, we show regional specificity for HF patterning and spatio-temporal control of the underlying molecular signals initiating the development of the pilosebaceous unit.

Differentiation of the sebaceous gland.

The sebaceous gland is renewed throughout adult life and homeostasis of this particular organ is controlled by a precise interplay of hormones, cytokines, signalling molecules and mediators of the lipid metabolism. Although the true function of sebaceous glands has yet to be fully determined, recent evidence demonstrated that normal homeostasis of the sebaceous gland and functional lipid metabolism of sebocytes is crucial for maintenance of the skin barrier. In addition, analysis of mutant mouse models revealed a close interdependency of the sebaceous gland with hair follicles because abnormal morphogenesis of sebaceous glands often results in degeneration of hair follicle structures. Anomalous regulation of sebaceous glands is involved in the pathogenesis of acne, one of the most prevalent human diseases, or could lead to formation of sebaceous hyperplasia and tumours. This review highlights some of the recent findings on the importance of signalling pathways controlling morphogenesis and differentiation of the sebaceous gland in vivo.