Carrie A. Ambler

Jagged 1 is a β-catenin target gene required for ectopic hair follicle formation in adult epidermis

The Wnt and Notch signalling pathways regulate hair follicle maintenance, but how they intersect is unknown. We show that Notch signalling is active in the hair follicle pre-cortex, a region of high Wnt activity, where commitment to hair lineages occurs. Deletion of jagged 1 (Jag1) results in inhibition of the hair growth cycle and conversion of hair follicles into cysts of cells undergoing interfollicular epidermal differentiation. Conversely, activation of Notch in adult epidermis triggers expansion of the base of the hair follicle, sebaceous gland enlargement and abnormal clumping of the follicles. In adult epidermis, the induction of new hair follicle formation by β-catenin is prevented by blocking Notch signalling pharmacologically or through Jag1 deletion. Conversely, activation of both pathways accelerates growth and differentiation of ectopic follicles.β -catenin stimulates Notch signalling by inducing Jag1 transcription. We conclude that the Notch pathway acts downstream of the Wnt/β-catenin pathway to determine epidermal cell fate.

Epidermal Notch signalling: differentiation, cancer and adhesion

The Notch pathway plays an important role in regulating epidermal differentiation. Notch ligands, receptors and effectors are expressed in a complex and dynamic pattern in embryonic and adult skin. Genetic ablation or activation of the pathway reveals that Notch signalling promotes differentiation of the hair follicle, sebaceous gland and interfollicular epidermal lineages and that Notch acts as an epidermal tumour suppressor. Notch signalling interacts with a range of other pathways to fulfil these functions and acts via RBP-Jκ dependent and independent mechanisms. The effects on differentiation can be cell autonomous and non-autonomous, and Notch contributes to stem cell clustering via modulation of cell adhesion.

The neural tube patterns vessels developmentally using the VEGF signaling pathway.

Embryonic blood vessels form in a reproducible pattern that interfaces with other embryonic structures and tissues, but the sources and identities of signals that pattern vessels are not well characterized. We hypothesized that the neural tube provides vascular patterning signal(s) that direct formation of the perineural vascular plexus (PNVP) that encompasses the neural tube at mid-gestation. Both surgically placed ectopic neural tubes and ectopic neural tubes engineered genetically were able to recruit a vascular plexus, showing that the neural tube is the source of a vascular patterning signal. In mouse-quail chimeras with the graft separated from the neural tube by a buffer of host cells, graft-derived vascular cells contributed to the PNVP, indicating that the neural tube signal(s) can act at a distance. Murine neural tube vascular endothelial growth factor A (VEGFA) expression was temporally and spatially correlated with PNVP formation, suggesting it is a component of the neural tube signal. A collagen explant model was developed in which presomitic mesoderm explants formed a vascular plexus in the presence of added VEGFA. Co-cultures between presomitic mesoderm and neural tube also supported vascular plexus formation, indicating that the neural tube could replace the requirement for VEGFA. Moreover, a combination of pharmacological and genetic perturbations showed that VEGFA signaling through FLK1 is a required component of the neural tube vascular patterning signal. Thus, the neural tube is the first structure identified as a midline signaling center for embryonic vascular pattern formation in higher vertebrates, and VEGFA is a necessary component of the neural tube vascular patterning signal. These data suggest a model whereby embryonic structures with little or no capacity for angioblast generation act as a nexus for vessel patterning.

Epidermal stem cells: location, potential and contribution to cancer

Epidermal stem cells have been classically characterized as slow‐cycling, long‐lived cells that reside in discrete niches in the skin. Gene expression studies of niche‐resident cells have revealed a number of stem cell markers and regulators, including the Wnt/β‐catenin, Notch, p63, c‐Myc and Hedgehog pathways. A new study challenges the traditional developmental paradigm of slow‐cycling stem cells and rapid‐cycling transit amplifying cells in some epidermal regions, and there is mounting evidence to suggest that multi‐lineage epidermal progenitors can be isolated from highly proliferative, non‐niche regions. Whether there is a unique microenvironment surrounding these progenitors remains to be determined. Interestingly, cancer stem cells derived from epidermal tumours exist independent of the classic skin stem cell niche, yet also have stem cell properties, including multi‐lineage differentiation. This review summarizes recent studies identifying the location and regulators of mouse and human epidermal stem cells and highlights the strategies used to identify cancer stem cells, including expression of normal epidermal stem cell markers, expression of cancer stem cell markers identified in other epidermal tumours and characterization of side‐population tumour cells. Copyright

Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin Model

Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification.

Stem cell-derived endothelial cells/progenitors migrate and pattern in the embryo using the VEGF signaling pathway

Endothelial precursor cells respond to molecular cues to migrate and assemble into embryonic blood vessels, but the signaling pathways involved in vascular patterning are not well understood. We recently showed that avian vascular patterning cues are recognized by mammalian angioblasts derived from somitic mesoderm through analysis of mouse-avian chimeras. To determine whether stem cell-derived endothelial cells/progenitors also recognize global patterning signals, murine ES cell-derived embryoid bodies (EBs) were grafted into avian hosts. ES cell-derived murine endothelial cells/progenitors migrated extensively and colonized the appropriate host vascular beds. They also formed mosaic vessels with avian endothelial cells. Unlike somite derived-endothelial cells, ES cell-derived endothelial cells/progenitors migrated across the host embryonic midline to the contralateral side. To determine the role of VEGF signaling in embryonic vascular patterning, EBs mutant for a VEGF receptor (flk-1(-/-)) or a signal (VEGF-A(-/-)) were grafted into quail hosts. Flk-1(-/-) EB grafts produced only rare endothelial cells that did not migrate or assemble into vessels. In contrast, VEGF-A(-/-) EB grafts produced endothelial cells that resembled wild-type and colonized host vascular beds, suggesting that host-derived signals can partially rescue mutant graft vascular patterning. VEGF-A(-/-) graft endothelial cells/progenitors crossed the host midline with much lower frequency than wild-type EB grafts, indicating that graft-derived VEGF compromised the midline barrier when present. Thus, ES cell-derived endothelial cells/progenitors respond appropriately to global vascular patterning cues, and they require the VEGF signaling pathway to pattern properly. Moreover, EB-avian chimeras provide an efficient way to screen mutations for vascular patterning defects.

Development of the Mouse Dermal Adipose Layer Occurs Independently of Subcutaneous Adipose Tissue and Is Marked by Restricted Early Expression of FABP4

The laboratory mouse is a key animal model for studies of adipose biology, metabolism and disease, yet the developmental changes that occur in tissues and cells that become the adipose layer in mouse skin have received little attention. Moreover, the terminology around this adipose body is often confusing, as frequently no distinction is made between adipose tissue within the skin, and so called subcutaneous fat. Here adipocyte development in mouse dorsal skin was investigated from before birth to the end of the first hair follicle growth cycle. Using Oil Red O staining, immunohistochemistry, quantitative RT-PCR and TUNEL staining we confirmed previous observations of a close spatio-temporal link between hair follicle development and the process of adipogenesis. However, unlike previous studies, we observed that the skin adipose layer was created from cells within the lower dermis. By day 16 of embryonic development (e16) the lower dermis was demarcated from the upper dermal layer, and commitment to adipogenesis in the lower dermis was signalled by expression of FABP4, a marker of adipocyte differentiation. In mature mice the skin adipose layer is separated from underlying subcutaneous adipose tissue by the panniculus carnosus. We observed that the skin adipose tissue did not combine or intermix with subcutaneous adipose tissue at any developmental time point. By transplanting skin isolated from e14.5 mice (prior to the start of adipogenesis), under the kidney capsule of adult mice, we showed that skin adipose tissue develops independently and without influence from subcutaneous depots. This study has reinforced the developmental link between hair follicles and skin adipocyte biology. We argue that because skin adipocytes develop from cells within the dermis and independently from subcutaneous adipose tissue, that it is accurately termed dermal adipose tissue and that, in laboratory mice at least, it represents a separate adipose depot.

Adult epidermal Notch activity induces dermal accumulation of T cells and neural crest derivatives through upregulation of jagged 1.

Notch signalling regulates epidermal differentiation and tumour formation via non-cell autonomous mechanisms that are incompletely understood. This study shows that epidermal Notch activation via a 4-hydroxy-tamoxifen-inducible transgene caused epidermal thickening, focal detachment from the underlying dermis and hair clumping. In addition, there was dermal accumulation of T lymphocytes and stromal cells, some of which localised to the blisters at the epidermal-dermal boundary. The T cell infiltrate was responsible for hair clumping but not for other Notch phenotypes. Notch-induced stromal cells were heterogeneous, expressing markers of neural crest, melanocytes, smooth muscle and peripheral nerve. Although Slug1 expression was expanded in the epidermis, the stromal cells did not arise through epithelial-mesenchymal transition. Epidermal Notch activation resulted in upregulation of jagged 1 in both epidermis and dermis. When Notch was activated in the absence of epidermal jagged 1, jagged 1 was not upregulated in the dermis, and epidermal thickening, blister formation, accumulation of T cells and stromal cells were inhibited. Gene expression profiling revealed that epidermal Notch activation resulted in upregulation of several growth factors and cytokines, including TNFα, the expression of which was dependent on epidermal jagged 1. We conclude that jagged 1 is a key mediator of non-cell autonomous Notch signalling in skin.

Epidermal Notch1 recruits RORγ+ group 3 innate lymphoid cells to orchestrate normal skin repair

Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ+ ILC3s into wounded dermis; RORγ+ ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ+ ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair.

Modified Methods for Growing 3-D Skin Equivalents: An Update

Artificial epidermis can be reconstituted in vitro by seeding primary epidermal cells (keratinocytes) onto a supportive substrate and then growing the developing skin equivalent at the air-liquid interface. In vitro skin models are widely used to study skin biology and for industrial drug and cosmetic testing. Here, we describe updated methods for growing 3-dimensional skin equivalents using de-vitalized, de-epidermalized dermis (DED) substrates including methods for DED substrate preparation, cell seeding, growth conditions, and fixation procedures.