Transient activaton of β-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours

When β-catenin signalling is disturbed from mid-gestation onwards lineage commitment is profoundly altered in postnatal mouse epidermis. We have investigated whether adult epidermis has the capacity for β-catenin-induced lineage conversion without prior embryonic priming. We fused N-terminally truncated, stabilised β-catenin to the ligand-binding domain of a mutant oestrogen receptor (ΔNβ -cateninER). ΔNβ-cateninER was expressed in the epidermis of transgenic mice under the control of the keratin 14 promoter and β -catenin activity was induced in adult epidermis by topical application of 4-hydroxytamoxifen (4OHT). Within 7 days of daily 4OHT treatment resting hair follicles were recruited into the hair growth cycle and epithelial outgrowths formed from existing hair follicles and from interfollicular epidermis. The outgrowths expressed Sonic hedgehog, Patched and markers of hair follicle differentiation, indicative of de novo follicle formation. The interfollicular epidermal differentiation program was largely unaffected but after an initial wave of sebaceous gland duplication sebocyte differentiation was inhibited. A single application of 4OHT was as effective as repeated doses in inducing new follicles and growth of existing follicles. Treatment of epidermis with 4OHT for 21 days resulted in conversion of hair follicles to benign tumours resembling trichofolliculomas. The tumours were dependent on continuous activation of β-catenin and by 28 days after removal of the drug they had largely regressed. We conclude that interfollicular epidermis and sebaceous glands retain the ability to be reprogrammed in adult life and that continuous β-catenin signalling is required to maintain hair follicle tumours.Peer reviewedFinal Published versio

Automated Identification and Measurement of Haematopoietic Stem Cells in 3D Intravital Microscopy Data

Image analysis and quantification of Haematopoietic stem cells (HSCs) position within their surrounding microenvironment in the bone marrow is a fast growing area of research, as it holds the key to understanding the dynamics of HSC-niche interactions and their multiple implications in normal tissue development and in response to various stress events. However, this area of research is very challenging due to the complex cellular structure of such images. Therefore, automated image analysis tools are required to simplify the biological interpretation of 3D HSC microenvironment images. In this chapter, we describe how 3D intravital microscopy data can be visualised and analysed using a computational method that allows the automated quantification of HSC position relative to surrounding niche components

Targeting adhesion to the vascular niche to improve therapy for acute myeloid leukemia

Niche hijack by malignant cells is considered to be a prominent cause of disease relapse. Barbier and colleagues uncover (E)-selectin as a novel mediator of malignant cell survival and regeneration which, upon blockade, has the potential to significantly improve therapeutic outcomes

The evolving view of the hematopoietic stem cell niche.

Hematopoietic stem cells (HSCs) reside in specialized microenvironments known as niches. The niche is essential to support HSC function and to maintain a correct balance between self-renewal and differentiation. Recent advances in defining different mesenchymal and endothelial bone marrow cell populations, as well as hematopoietic stem and progenitor cells, greatly enhanced our understanding of these niches and of the molecular mechanisms by which they regulate HSC function. In addition to the role in maintaining HSC homeostasis, the niche has also been implicated in the pathogenesis of blood disorders including hematological malignancies. Characterizing the extrinsic regulators and the cellular context in which the niches interact with HSCs will be crucial to define new strategies to enhance blood regeneration. Furthermore, a better understanding of the role of the niche in leukemia development will open new possibilities for the treatment of these disorders by using therapies aiming to target the leukemic niche specifically. To update on recent findings on this topic, the International Society for Experimental Hematology (ISEH) organized a webinar, presented by Prof. Sean J. Morrison and Dr. Simón Méndez-Ferrer and moderated by Dr. Cristina Lo Celso, entitled "The evolving view of the hematopoietic stem cell niche," which we summarize here

Effect of the CXCR4 antagonist plerixafor on endogenous neutrophil dynamics in the bone marrow, lung and spleen

Treatment with the CXCR4 antagonist, plerixafor (AMD3100), has been proposed for clinical use in patients with WHIM (warts, hypogammaglobulinemia, infections and myelokathexis) syndrome and in pulmonary fibrosis. However, there is controversy with respect to the impact of plerixafor on neutrophil dynamics in the lung, which may affect its safety profile. In this study we investigated the kinetics of endogenous neutrophils by direct imaging, using confocal intravital microscopy in mouse bone marrow, spleen and lungs. Neutrophils are observed increasing their velocity and exiting the bone marrow following plerixafor administration, with a concomitant increase in neutrophil numbers in the blood and spleen, while the marginated pool of neutrophils in the lung microvasculature remained unchanged in terms of numbers and cell velocity. Use of autologous radiolabeled neutrophils and SPECT/CT imaging in healthy volunteers showed that plerixafor did not affect GM-CSF-primed neutrophil entrapment or release in the lungs. Taken together these data suggest that plerixafor causes neutrophil mobilization from the bone marrow but does not impact on lung marginated neutrophil dynamics and thus is unlikely to compromise respiratory host defense both in humans and mice.This work was funded by a grant provided to JP by the Lung Foundation Netherlands (5.2.14.058JO), the NIHR Cambridge Biomedical Research Centre and NIHR Imperial Biomedical Research Centre. ERC and CS’ laboratories receive grant support from the Medical Research Council, Wellcome Trust, NIHR, GlaxoSmithKline, MedImmune Ltd., and Bristol-Myers Squibb. CLC is supported by Bloodwise (12033), CRUK (C36195/A1183) and European Research Council (ERC) (337066). CP is supported by Bloodwise (12033). The Facility for Imaging by Light Microscopy (FILM) at Imperial College London is part-supported by funding from the Wellcome Trust (grant 104931/Z/14/Z) and BBSRC (grant BB/L015129/1). KDF is supported by funding from the Wellcome Trust (201356/Z/16/Z). LMC is supported by core funding from Cancer Research UK (A23983 and A17196)

Tracking Single Cells in Live Animals Using a Photoconvertible Near-Infrared Cell Membrane Label

We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4+ T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution

Guanine Nucleotide Exchange Factor Vav1 Regulates Perivascular Homing and Bone Marrow Retention of Hematopoietic Stem and Progenitor Cells

Engraftment and maintenance of hematopoietic stem and progenitor cells (HSPC) depend on their ability to respond to extracellular signals from the bone marrow microenvironment, but the critical intracellular pathways integrating these signals remain poorly understood. Furthermore, recent studies provide contradictory evidence of the roles of vascular versus osteoblastic niche components in HSPC function. To address these questions and to dissect the complex upstream regulation of Rac GTPase activity in HSPC, we investigated the role of the hematopoietic-specific guanine nucleotide exchange factor Vav1 in HSPC localization and engraftment. Using intravital microscopy assays, we demonstrated that transplanted $Vav1^{−/−}$ HSPC showed impaired early localization near $nestin^+$ perivascular mesenchymal stem cells; only 6.25% of $Vav1^{−/−}$ HSPC versus 45.8% of wild-type HSPC were located less than 30 μm from a $nestin^+$ cell. Abnormal perivascular localization correlated with decreased retention of $Vav1^{−/−}$ HSPC in the bone marrow (44–60% reduction at 48 h posttransplant, compared with wild-type) and a very significant defect in short- and long-term engraftment in competitive and noncompetitive repopulation assays (<1.5% chimerism of $Vav1^{−/−}$ cells vs. 53–63% for wild-type cells). The engraftment defect of $Vav1^{−/−}$ HSPC was not related to alterations in proliferation, survival, or integrin-mediated adhesion. However, $Vav1^{−/−}$ HSPC showed impaired responses to $SDF1\alpha$, including reduced in vitro migration in time-lapse microscopy assays, decreased circadian and pharmacologically induced mobilization in vivo, and dysregulated Rac/Cdc42 activation. These data suggest that Vav1 activity is required specifically for $SDF1\alpha$-dependent perivascular homing of HSPC and suggest a critical role for this localization in retention and subsequent engraftment

Haematopoietic focal adhesion kinase deficiency alters haematopoietic homeostasis to drive tumour metastasis

Metastasis is the main cause of cancer-related death and thus understanding the molecular and cellular mechanisms underlying this process is critical. Here, our data demonstrate, contrary to established dogma, that loss of haematopoietic-derived focal adhesion kinase (FAK) is sufficient to enhance tumour metastasis. Using both experimental and spontaneous metastasis models, we show that genetic ablation of haematopoietic FAK does not affect primary tumour growth but enhances the incidence of metastasis significantly. At a molecular level, haematopoietic FAK deletion results in an increase in PU-1 levels and decrease in GATA-1 levels causing a shift of hematopoietic homeostasis towards a myeloid commitment. The subsequent increase in circulating granulocyte number, with an increase in serum CXCL12 and granulocyte CXCR4 levels, was required for augmented metastasis in mice lacking haematopoietic FAK. Overall our findings provide a mechanism by which haematopoietic FAK controls cancer metastasis

Systematic tracking of altered haematopoiesis during sporozoite-mediated malaria development reveals multiple response points.

Haematopoiesis is the complex developmental process that maintains the turnover of all blood cell lineages. It critically depends on the correct functioning of rare, quiescent haematopoietic stem cells (HSCs) and more numerous, HSC-derived, highly proliferative and differentiating haematopoietic progenitor cells (HPCs). Infection is known to affect HSCs, with severe and chronic inflammatory stimuli leading to stem cell pool depletion, while acute, non-lethal infections exert transient and even potentiating effects. Both whether this paradigm applies to all infections and whether the HSC response is the dominant driver of the changes observed during stressed haematopoiesis remain open questions. We use a mouse model of malaria, based on natural, sporozoite-driven Plasmodium berghei infection, as an experimental platform to gain a global view of haematopoietic perturbations during infection progression. We observe coordinated responses by the most primitive HSCs and multiple HPCs, some starting before blood parasitaemia is detected. We show that, despite highly variable inter-host responses, primitive HSCs become highly proliferative, but mathematical modelling suggests that this alone is not sufficient to significantly impact the whole haematopoietic cascade. We observe that the dramatic expansion of Sca-1(+) progenitors results from combined proliferation of direct HSC progeny and phenotypic changes in downstream populations. We observe that the simultaneous perturbation of HSC/HPC population dynamics is coupled with early signs of anaemia onset. Our data uncover a complex relationship between Plasmodium and its host's haematopoiesis and raise the question whether the variable responses observed may affect the outcome of the infection itself and its long-term consequences on the host