Elizabeth Clayton

A single type of progenitor cell maintains normal epidermis

According to the current model of adult epidermal homeostasis, skin tissue is maintained by two discrete populations of progenitor cells: self-renewing stem cells; and their progeny, known as transit amplifying cells, which differentiate after several rounds of cell division. By making use of inducible genetic labelling, we have tracked the fate of a representative sample of progenitor cells in mouse tail epidermis at single-cell resolution in vivo at time intervals up to one year. Here we show that clone-size distributions are consistent with a new model of homeostasis involving only one type of progenitor cell. These cells are found to undergo both symmetric and asymmetric division at rates that ensure epidermal homeostasis. The results raise important questions about the potential role of stem cells on tissue maintenance in vivo.

A Crucial Role for the p110δ Subunit of Phosphatidylinositol 3-Kinase in B Cell Development and Activation

Mice lacking the p110δ catalytic subunit of phosphatidylinositol 3-kinase have reduced numbers of B1 and marginal zone B cells, reduced levels of serum immunoglobulins, respond poorly to immunization with type II thymus-independent antigen, and are defective in their primary and secondary responses to thymus-dependent antigen. p110δ−/− B cells proliferate poorly in response to B cell receptor (BCR) or CD40 signals in vitro, fail to activate protein kinase B, and are prone to apoptosis. p110δ function is required for BCR-mediated calcium flux, activation of phosphlipaseCγ2, and Brutons tyrosine kinase. Thus, p110δ plays a critical role in B cell homeostasis and function.

Signal transduction through Vav-2 participates in humoral immune responses and B cell maturation.

B and T lymphocytes develop normally in mice lacking the guanine nucleotide exchange factor Vav-2. However, the immune responses to type II thymus-independent antigen as well as the primary response to thymus-dependent (TD) antigen are defective. Vav-2–deficient mice are also defective in their ability to switch immunoglobulin class, form germinal centers and generate secondary immune responses to TD antigens. Mice lacking both Vav-1 and Vav-2 contain reduced numbers of B lymphocytes and display a maturational block in the development of mature B cells. B cells from Vav-1−/−Vav-2−/− mice respond poorly to antigen receptor triggering, both in terms of proliferation and calcium release. These studies show the importance of Vav-2 in humoral immune responses and B cell maturation.

Characterisation of a stereotypical cellular and extracellular adult liver progenitor cell niche in rodents and diseased human liver

Background Stem/progenitor cell niches in tissues regulate stem/progenitor cell differentiation and proliferation through local signalling. Objective To examine the composition and formation of stem progenitor cell niches. Methods The composition of the hepatic progenitor cell niche in independent models of liver injury and hepatic progenitor cell activation in rodents and humans was studied. To identify the origin of the progenitor and niche cells, sex-mismatched bone marrow transplants in mice, who had received the choline–ethionine-deficient-diet to induce liver injury and progenitor cell activation, were used. The matrix surrounding the progenitor cells was described by immunohistochemical staining and its functional role controlling progenitor cell behaviour was studied in cell culture experiments using different matrix layers. Results The progenitor cell response in liver injury is intimately surrounded by myofibroblasts and macrophages, and to a lesser extent by endothelial cells. Hepatic progenitor cells are not of bone marrow origin; however, bone marrow-derived cells associate intimately with these cells and are macrophages. Laminin always surrounds the progenitor cells. In vitro studies showed that laminin aids maintenance of progenitor and biliary cell phenotype and promotes their gene expression (Dlk1, Aquaporin 1, γGT) while inhibiting hepatocyte differentiation and gene expression (CEPB/α). Conclusions During liver damage in rodents and humans a stereotypical cellular and laminin niche forms around hepatic progenitor cells. Laminin helps maintenance of undifferentiated progenitor cells. The niche links the intrahepatic progenitor cells with bone marrow-derived cells and links tissue damage with progenitor cell-mediated tissue repair.

Vav-2 controls NFAT-dependent transcription inB- but not T-lymphocytes

We show here that Vav‐2 is tyrosine phosphorylated following antigen receptor engagement in both B‐ and T‐cells, but potentiates nuclear factor of activated T cells (NFAT)‐dependent transcription only in B cells. Vav‐2 function requires the N‐terminus, as well as functional Dbl homology and SH2 domains. More over, the enhancement of NFAT‐dependent transcription by Vav‐2 can be inhibited by a number of dominant‐negative GTPases. The ability of Vav‐2 to potentiate NFAT‐dependent transcription correlates with its ability to promote a sustained calcium flux. Thus, Vav‐2 augments the calcium signal in B cells but not T cells, and a truncated form of Vav‐2 can neither activate NFAT nor augment calcium signaling. The CD19 co‐receptor physically interacts with Vav‐2 and synergistically enhances Vav‐2 phosphorylation induced by the B‐cell receptor (BCR). In addition, we found that Vav‐2 augments CD19‐stimulated NFAT‐ dependent transcription, as well as transcription from the CD5 enhancer. These data suggest a role for Vav‐2 in transducing BCR signals to the transcription factor NFAT and implicate Vav‐2 in the integration of BCR and CD19 signaling.

Vav-Dependent and Vav-Independent Phosphatidylinositol 3-Kinase Activation in Murine B Cells Determined by the Nature of the Stimulus

We show in this study that B cell activation following high avidity ligation of IgM or coligation of membrane Ig with CD19 elicits similar levels of Ca2+ flux using different mechanisms. Each form of activation requires the function of Vav and PI3K. However, Vav regulates Ca2+ flux independently of PI3K following anti-IgM cross-linking. By contrast, Vav function is essential for PI3K activation following membrane Ig (mIg)/CD19 coligation. Inhibition of PI3K revealed anti-IgM-stimulated Ca2+ flux has a PI3K-independent component, while Ca2+ flux following mIg/CD19 coligation is totally PI3K dependent. The p85α and p110δ subunits of PI3K both participate in anti-IgM and mIg/CD19 coligation-induced Ca2+ flux, although the defects are not as severe as observed after pharmacological inhibition. This may reflect the recruitment of additional PI3K subunits, as we found that p110α becomes associated with CD19 upon B cell activation. These data show that the nature of the Ag encountered by B cells determines the contribution of Vav proteins to PI3K activation. Our results indicate that the strong signals delivered by multivalent cross-linking agents activate B cells in a qualitatively different manner from those triggered by coreceptor recruitment.

The isolation and in vitro expansion of hepatic Sca-1 progenitor cells.

The intra-hepatic population of liver progenitor cells expands during liver injury when hepatocyte proliferation is inhibited. These cells can be purified by density gradient centrifugation and cultured. Separated by size only this population contains small cells of hematopoietic, epithelial and endothelial lineages and is thought to contain liver stem cells. The identity of liver stem cells remains unknown although there is some evidence that tissue Sca1(+) CD45(-) cells display progenitor cell characteristics. We identified both intra-hepatic and gall bladder Sca1(+) cells following liver injury and expanded ex vivo Sca1 cells as part of heterogenous cell culture or as a purified population. We found significant difference between the proliferation of Sca-1 cells when plated on laminin or collagen I while proliferation of heterogenous population was not affected by the extracellular matrix indicating the necessity for culture of Sca1(+) cells with laminin matrix or laminin producing cells in long term liver progenitor cell cultures.

Differential labelling of bulk endocytosis in nerve terminals by FM dyes

Bulk endocytosis is triggered in central nerve terminals during intense physiological stimulation. This endocytosis pathway can be labelled by the dye FM1-43 but not its more hydrophilic counterpart FM2-10. This selective labelling was proposed to be due to the retention of FM1-43, but not FM2-10, in slowly retrieving structures after washout of the dye. However, this explanation assumed that bulk endocytosis was a slow process that persisted after stimulation. We have recently shown that the great majority of bulk endocytosis occurs during stimulation, therefore another explanation for the specific labelling of this pathway by FM1-43 must be found. In this paper we show that the ability of FM dyes to label bulk endocytosis is dependent on the concentration of dye used and not their washout properties. When the loading concentration of FM1-43 was reduced 10-fold, its ability to label bulk endocytosis was lost. Conversely when the loading concentration of FM2-10 was increased 10-fold, it now labelled the pathway. This suggests that a difference in affinity of bulk endosome membranes for FM1-43 and FM2-10 underlies the disparity in labelling.

BCR activation of PI3K is Vav-independent in murine B cells

BCR (B-cell antigen receptor)-induced Ca(2+) signalling is initiated by activation of tyrosine kinases, which in concert with adaptor proteins and lipid kinases regulate PLC (phospholipase C) gamma2 activation. Vav and PI3K (phosphoinositide 3-kinase) are required for optimal Ca(2+) responses, although it has not been established, in primary B-cells, if both proteins are components of the same pathway. In vitro evidence suggests that binding of the PI3K lipid product PIP3 to Vav pleckstrin homology domain contributes to Vav activation. However, pharmacological inhibition of PI3K by wortmannin or deletion of the p110delta catalytic subunit has no effect on Vav activation in response to BCR engagement, suggesting that this mechanism does not operate in vivo. We also show that PI3K recruitment to phosphorylated-tyrosine-containing complexes is Vav-independent. Taken together with our previous observation that protein kinase B phosphorylation is normal in Vav-deficient B-cells, we suggest that PI3K activation is Vav-independent in response to strong signals delivered by multivalent cross-linking.