Fibroblasts are the major mesenchymal cell type in connective tissue and deposit the collagen and elastic fibers of the extracellular matrix (ECM)1. Even within a single tissue fibroblasts exhibit remarkable functional diversity, but it is not known whether this reflects the existence of a differentiation hierarchy or is a response to different environmental factors. Here we show, using transplantation assays and lineage tracing, that the fibroblasts of skin connective tissue arise from two distinct lineages. One forms the upper dermis, including the dermal papilla that regulates hair growth and the arrector pili muscle (APM), which controls piloerection. The other forms the lower dermis, including the reticular fibroblasts that synthesise the bulk of the fibrillar ECM, and the pre-adipocytes and adipocytes of the hypodermis. The upper lineage is required for hair follicle formation. In wounded adult skin, the initial wave of dermal repair is mediated by the lower lineage and upper dermal fibroblasts are recruited only during re-epithelialisation. Epidermal beta-catenin activation stimulates expansion of the upper dermal lineage, rendering wounds permissive for hair follicle formation. Our findings explain why wounding is linked to formation of ECM-rich scar tissue that lacks hair follicles2-4. They also form a platform for discovering fibroblast lineages in other tissues and for examining fibroblast changes in ageing and disease.

Adult stem cells are characterized by self-renewal and multilineage differentiation, and these properties seem to be regulated by signals from adjacent differentiated cell types and by extracellular matrix molecules, which collectively define the stem cell "niche." Self-renewal is essential for the lifelong persistence of stem cells, but its regulation is poorly understood. In the mammalian brain, neurogenesis persists in two germinal areas, the subventricular zone (SVZ) and the hippocampus, where continuous postnatal neuronal production seems to be supported by neural stem cells (NSCs). Here we show that pigment epithelium-derived factor (PEDF) is secreted by components of the murine SVZ and promotes self-renewal of adult NSCs in vitro. In addition, intraventricular PEDF infusion activated slowly dividing stem cells, whereas a blockade of endogenous PEDF decreased their cycling. These data demonstrate that PEDF is a niche-derived regulator of adult NSCs and provide evidence for a role for PEDF protein in NSC maintenance.

The gene for the atypical Notch ligand Delta-like homologue 1 (Dlk1) encodes membrane-bound and secreted isoforms functioning in multiple developmental processes in vitro and in vivo. Dlk1, a member of a cluster of imprinted genes, is expressed from the paternally-inherited chromosome1,2. Here we show that mice deficient in Dlk1 exhibit defects in postnatal neurogenesis within the subventricular zone (SVZ), a developmental continuum resulting in depletion of mature neurons in the olfactory bulb. We show that DLK1 is a factor secreted by niche-astrocytes, while its membrane-bound isoform is present in neural stem cells (NSCs) being required for the inductive effect of secreted DLK1 on self-renewal. Surprisingly, we find a requirement for Dlk1 expressed from both maternal and paternally inherited chromosomes. Selective absence of Dlk1 imprinting in both NSCs and niche astrocytes is associated with postnatal acquisition of DNA methylation at the germ line-derived imprinting control region (IG-DMR). The results emphasize molecular relationships between NSCs and niche-astrocytes identifying a signalling system coded by a single gene functioning co-ordinately in both cell types. The modulation of genomic imprinting in a stem cell environment adds a new level of epigenetic regulation to the establishment and maintenance of the niche raising wider questions about the adaptability, function, and evolution of imprinting within specific developmental contexts.

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Proliferation in the subependymal zone (SEZ) and neurogenesis in the olfactory bulb decline in the forebrain of telomerase-deficient mice. The present work reveals additional effects of telomere shortening on neuronal differentiation, as adult multipotent progenitors with critically short telomeres yield reduced numbers of neurons that, furthermore, exhibit underdeveloped neuritic arbors. Genetic data indicate that the tumor suppressor protein p53 not only mediates the adverse effects of telomere attrition on proliferation and selfrenewal but it is also involved in preventing normal neuronal differentiation of adult progenitors with dysfunctional telomeres. Interestingly, progenitor cells with short telomeres obtained from fetal brains do not exhibit any replicative defects but also fail to acquire a fully mature neuritic arbor, demonstrating cell cycle-independent effects of telomeres on neuronal differentiation. The negative effect of p53 on neuritogenesis is mechanistically linked to its cooperation with the Notch pathway in the upregulation of small GTPase RhoA kinases, Rock1 and Rock2, suggesting a potential link between DNA damage and the Notch signaling pathway in the control of neuritogenesis. We also show that telomerase expression is downregulated in the SEZ of aging mice leading to telomere length reductions in neurosphere-forming cells and deficient neurogenesis and neuritogenesis. Our results suggest that age-related deficits could be caused partly by dysfunctional telomeres and demonstrate that p53 is a central modulator of adult neurogenesis, regulating both the production and differentiation of postnatally generated olfactory neurons.

Interactions of adult neural stem cells (NSCs) with supportive vasculature appear critical for their maintenance and function, although the molecular details are still under investigation. Neurotrophin (NT)-3 belongs to the NT family of trophic factors, best known for their effects in promoting neuronal survival. Here we show that NT-3 produced and secreted by endothelial cells of brain and choroid plexus capillaries is required for the quiescence and long-term maintenance of NSCs in the mouse subependymal niche. Uptake of NT-3 from irrigating vasculature and cerebrospinal fluid (CSF) induces the rapid phosphorylation of endothelial nitric oxide (NO) synthase present in the NSCs, leading to the production of NO, which subsequently acts as a cytostatic factor. Our results identify a novel interaction between stem cells and vasculature/CSF compartments that is mediated by an unprecedented role of a neurotrophin and indicate that stem cells can regulate their own quiescence in response to endothelium-secreted molecules.

Two known germinal zones continue to generate new neurons and glia in the adult mammalian brain: the subventricular zone (SVZ), lining the lateral walls of the lateral ventricle, and the subgranular zone of the dentate gyrus. Here we describe a region we will refer to as the subcallosal zone (SCZ). The SCZ is a caudal extension of the SVZ that is no longer associated to an open ventricle. It lies between the hippocampus and the corpus callosum. Cells isolated from the SCZ and cultured as neurospheres behave as neural stem cells in vitro. Using electron and light microscopy, we describe the cell types present in this region and how their organization differs from that of the SVZ. Using retroviral labeling and homotypic-homochronic microtransplantation techniques, we show that the majority of cells born in the SCZ migrate into the corpus callosum to become oligodendrocytes in vivo. This study defines the organization and fate of cells born in a large germinal region of the adult forebrain.

We describe a protocol developed/modified by our group for the ex vivo and in vivo assessment of the response to a soluble factor of murine neural stem cells from the adult sub-ventricular zone (SVZ). The procedure includes several experimental options that can be used either independently or in combination. Potential factor effects on self-renewal, survival and proliferation are assayed by means of neurosphere cultures, with the factor administered directly in vitro to the culture plates (Step 1) or infused in vivo immediately before tissue dissociation (Step 3). We also use bromodeoxiuridine (BrdU) retention to label slowly dividing cells in vivo and subsequently perform two different types of experiments. In one set of experiments, the factor is added to primary cultures of stem cells obtained from the BrdU-pulsed animals and effects are tested on label-retaining cells after immunocytochemistry (Step 2). In another set, prolonged intraventricular infusion of the factor in BrdU-pulsed animals is followed by immunohistochemical analysis of BrdU labeling in the intact SVZ (Step 4). The minimum estimated time for the full combined procedure is 45 d.