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 Jag1transcription. We conclude that the Notch pathway acts downstream of the Wnt/β-catenin pathway to determine epidermal cell fate.

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.

Mice lacking the vascular endothelial growth factor (VEGF) receptor flt-1 die of vascular overgrowth, and we are interested in how flt-1 normally prevents this outcome. Our results support a model whereby aberrant endothelial cell division is the cellular mechanism resulting in vascular overgrowth, and they suggest that VEGF-dependent endothelial cell division is normally finely modulated by flt-1 to produce blood vessels. Flt-1 ؊/؊ embryonic stem cell cultures had a 2-fold increase in endothelial cells by day 8, and the endothelial cell mitotic index was significantly elevated before day 8. . Some of the mitogenic signals that promote division of endothelial cells and their precursors are known, but how these signals are modulated to initiate cell divisions only when and where they are needed is not known in detail. After blood vessels initially form, maturation and remodeling steps involve the recruitment of ancillary cells, such as smooth muscle and pericytes. These cells and the extracellular matrix that is also produced can negatively modulate endothelial cell division. [4][5][6][7][8] However, modulators of endothelial cell mitogenesis at the earliest stages of blood vessel formation have not been identified.The vascular endothelial growth factor (VEGF) signaling pathway is clearly critical to both early endothelial cell division and morphogenesis, and its regulation is complex (reviewed in Ferrara & Davis-Smyth 9 and Neufeld et al 10 ). Mouse embryos lacking even one copy of the VEGF gene die in utero with severe vascular defects, and vascular development in differentiating embryonic stem (ES) cells is compromised in VEGF-A ϩ/Ϫ and VEGF-A Ϫ/Ϫ ES cells in a dose-dependent manner. [11][12][13] Moreover, even modestly elevated levels of VEGF lead to vascular abnormalities, 14 and large doses of VEGF invariably severely compromise both vascular development and neovascularization in adult organisms. [15][16][17] These findings suggest that VEGF signaling must be precisely controlled during vascularization to result in proper vessels. The location and duration of VEGF expression provide the first level of control, [18][19][20][21] but other components of the pathway are likely to be involved in fine-tuning the signal.Two high-affinity receptors, flk-1 and flt-1, participate in VEGF signal transduction and are candidates to be involved in fine-tuning mechanisms. Both receptors are membrane-spanning receptor tyrosine kinases that bind VEGF with high affinity, 22-26 but their effects on VEGF signaling are very different. Mice or ES cells lacking flk-1 have little or no blood vessel formation, suggesting that many downstream effects of VEGF on endothelial cells are mediated through flk-1. 27,28 Specifically, numerous studies show that VEGF signaling through flk-1 produces a strong mitogenic signal for endothelial cells. [29][30][31][32] In contrast, VEGF binding to flt-1 does not produce a strong mitogenic signal, and flt-1 Ϫ/Ϫ mice die at mid-gestation with vascular overgrowth and disorganization. 23,29,33 This ph...

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.

Vascular development requires the assembly of precursor cells into blood vessels, but how embryonic vessels are assembled is not well understood. To determine how vascular cells migrate and assemble into vessels of the trunk and limb, marked somite-derived angioblasts were followed in developing embryos. Injection of avian somites with the cell-tracker DiI showed that somite-derived angioblasts in unperturbed embryos migrated extensively and contributed to trunk and limb vessels. Mouse-avian chimeras with mouse presomitic mesoderm grafts had graft-derived endothelial cells in blood vessels at significant distances from the graft, indicating that mouse angioblasts migrated extensively in avian hosts. Mouse graft-derived endothelial cells were consistently found in trunk vessels, such as the perineural vascular plexus, the cardinal vein, and presumptive intersomitic vessels, as well as in vessels of the limb and kidney rudiment. This reproducible pattern of graft colonization suggests that avian vascular patterning cues for trunk and limb vessels are recognized by mammalian somitic angioblasts. Mouse-quail chimeras stained with both the quail vascular marker QH1 and the mouse vascular marker PECAM-1 had finely chimeric vessels, with graft-derived mouse cells interdigitated with quail vascular cells in most vascular beds colonized by graft cells. Thus, diverse trunk and limb blood vessels have endothelial cells that developed from migratory somitic angioblasts, and assembly of these vessels is likely to have a large vasculogenic component.

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.

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.

We recently reported that the peptide C-K4-M2GlyR mimics the action of chloride channels when incorporated into the apical membrane of cultured renal epithelial monolayers. C-K4-M2GlyR is one of a series of peptides that were prepared by the addition of lysine residues to the N- or C-terminus of the M2 transmembrane sequence of the brain glycine receptor. This study addresses how such modifications affect physical properties such as aqueous solubility, aggregation, and secondary structure, as well as the ability of the modified peptides to form channels in epithelial monolayers. A graded improvement in solubility with a concomitant decrease in aggregation in aqueous media was observed for the M2GlyR transmembrane sequences. Increases in short-circuit current (I(SC)) of epithelial monolayers were observed after treatment with some but not all of the peptides. The bioactivity was higher for the more soluble, less aggregated M2GlyR peptides. As described in our previous communication, sensitivity of channel activity to diphenylamine-2-carboxylate, a chloride channel blocker, and bumetanide, an inhibitor of the Na/K/2Cl cotransporter, was used to assess changes in chloride selectivity for the different assembled channel-forming peptides. The unmodified M2GlyR sequence and the modified peptides with less positive charge are more sensitive to these agents than are the more highly charged forms. This study shows that relatively insoluble transmembrane sequences can be modified such that they are easier to purify and deliver in the absence of organic solvents with retention of membrane association, insertion, and assembly.