Gata3 targets Runx1 in the embryonic haematopoietic stem cell niche.

Runx1 is an important haematopoietic transcription factor as stressed by its involvement in a number of haematological malignancies. Furthermore, it is a key regulator of the emergence of the first haematopoietic stem cells (HSCs) during development. The transcription factor Gata3 has also been linked to haematological disease and was shown to promote HSC production in the embryo by inducing the secretion of important niche factors. Both proteins are expressed in several different cell types within the aorta-gonads-mesonephros (AGM) region, in which the first HSCs are generated; however, a direct interaction between these two key transcription factors in the context of embryonic HSC production has not formally been demonstrated. In this current study, we have detected co-localisation of Runx1 and Gata3 in rare sub-aortic mesenchymal cells in the AGM. Furthermore, the expression of Runx1 is reduced in Gata3 -/- embryos, which also display a shift in HSC emergence. Using an AGM-derived cell line as a model for the stromal microenvironment in the AGM and performing ChIP-Seq and ChIP-on-chip experiments, we demonstrate that Runx1, together with other key niche factors, is a direct target gene of Gata3. In addition, we can pinpoint Gata3 binding to the Runx1 locus at specific enhancer elements which are active in the microenvironment. These results reveal a direct interaction between Gata3 and Runx1 in the niche that supports embryonic HSCs and highlight a dual role for Runx1 in driving the transdifferentiation of haemogenic endothelial cells into HSCs as well as in the stromal cells that support this process.This work was supported by an Intermediate Fellowship (K.O.) and a Junior Fellowship (S.R.F.) from the Kay Kendall Leukaemia Fund, a British Society for Haematology Early Stage Investigator Fellowship (K.O.) as well as funding from Bloodwise (N.K.W. and B.G.), MRC (N.K.W. and B.G.) and the Wellcome Trust (N.K.W. and B.G.). MdB is funded by a programme in the MRC Molecular Hematology Unit Core award (Grant number: MC_UU_12009/2). Core facilities are supported by Strategic Award WT100140, equipment grant 093026 and centre grant MR/K017047/1

Mll-AF4 Confers Enhanced Self-Renewal and Lymphoid Potential during a Restricted Window in Development.

MLL-AF4+ infant B cell acute lymphoblastic leukemia is characterized by an early onset and dismal survival. It initiates before birth, and very little is known about the early stages of the disease's development. Using a conditional Mll-AF4-expressing mouse model in which fusion expression is targeted to the earliest definitive hematopoietic cells generated in the mouse embryo, we demonstrate that Mll-AF4 imparts enhanced B lymphoid potential and increases repopulation and self-renewal capacity during a putative pre-leukemic state. This occurs between embryonic days 12 and 14 and manifests itself most strongly in the lymphoid-primed multipotent progenitor population, thus pointing to a window of opportunity and a potential cell of origin. However, this state alone is insufficient to generate disease, with the mice succumbing to B cell lymphomas only after a long latency. Future analysis of the molecular details of this pre-leukemic state will shed light on additional events required for progression to acute leukemia.Core facilities at the Cambridge Institute for Medical Research are supported by Strategic Award WT100140 and equipment grant 093026; core facilities at the Edinburgh MRC Centre for Regenerative Medicine are supported by centre grant MR/K017047/1. This work was funded by a Bloodwise Bennett Senior Fellowship (10015 to K.O.), a Wellcome Trust Clinical PhD Studentship (097454/z/11/z to N.A.B.) the Gabrielle’s Angel Foundation for Cancer Research (to K.O.), and the Kay Kendall Leukaemia Fund (to K.O.).This is the final version of the article. It first appeared from Cell Press/Elsevier at http://dx.doi.org/10.1016/j.celrep.2016.06.046

DNA damage signalling from the placenta to foetal blood as a potential mechanism for childhood leukaemia initiation

Abstract For many diseases with a foetal origin, the cause for the disease initiation remains unknown. Common childhood acute leukaemia is thought to be caused by two hits, the first in utero and the second in childhood in response to infection. The mechanism for the initial DNA damaging event are unknown. Here we have used in vitro, ex vivo and in vivo models to show that a placental barrier will respond to agents that are suspected of initiating childhood leukaemia by releasing factors that cause DNA damage in cord blood and bone marrow cells, including stem cells. We show that DNA damage caused by in utero exposure can reappear postnatally after an immune challenge. Furthermore, both foetal and postnatal DNA damage are prevented by prenatal exposure of the placenta to a mitochondrially-targeted antioxidant. We conclude that the placenta might contribute to the first hit towards leukaemia initiation by bystander-like signalling to foetal haematopoietic cells

Identification of the skeletal progenitor cells forming osteophytes in osteoarthritis.

OBJECTIVES: Osteophytes are highly prevalent in osteoarthritis (OA) and are associated with pain and functional disability. These pathological outgrowths of cartilage and bone typically form at the junction of articular cartilage, periosteum and synovium. The aim of this study was to identify the cells forming osteophytes in OA. METHODS: Fluorescent genetic cell-labelling and tracing mouse models were induced with tamoxifen to switch on reporter expression, as appropriate, followed by surgery to induce destabilisation of the medial meniscus. Contributions of fluorescently labelled cells to osteophytes after 2 or 8 weeks, and their molecular identity, were analysed by histology, immunofluorescence staining and RNA in situ hybridisation. Pdgfrα-H2BGFP mice and Pdgfrα-CreER mice crossed with multicolour Confetti reporter mice were used for identification and clonal tracing of mesenchymal progenitors. Mice carrying Col2-CreER, Nes-CreER, LepR-Cre, Grem1-CreER, Gdf5-Cre, Sox9-CreER or Prg4-CreER were crossed with tdTomato reporter mice to lineage-trace chondrocytes and stem/progenitor cell subpopulations. RESULTS: Articular chondrocytes, or skeletal stem cells identified by Nes, LepR or Grem1 expression, did not give rise to osteophytes. Instead, osteophytes derived from Pdgfrα-expressing stem/progenitor cells in periosteum and synovium that are descendants from the Gdf5-expressing embryonic joint interzone. Further, we show that Sox9-expressing progenitors in periosteum supplied hybrid skeletal cells to the early osteophyte, while Prg4-expressing progenitors from synovial lining contributed to cartilage capping the osteophyte, but not to bone. CONCLUSION: Our findings reveal distinct periosteal and synovial skeletal progenitors that cooperate to form osteophytes in OA. These cell populations could be targeted in disease modification for treatment of OA

A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise.

The autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sympathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. Here, we describe that early postnatally, a subset of sympathetic nerve fibers undergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic dependency mediated through GDNF-family receptor-α2 (GFRα2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innervation for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-dependent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innervation reduces osteocyte survival and function, causing osteopenia and impaired skeletal adaptation to moderate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects

Dll-4 and IL-7 are up-regulated by three-dimensional cultured keratinocytes.

<p>(A) Dll-4 gene expression is strongly up-regulated in 3D cultured Hacat keratinocytes either alone or in the presence of fibroblasts. The differences between 3D and 2D either Hacat (*p<0.01) or cocultures (**p<0.05) are significant. No differences were observed in the housekeeping gene expression in all the conditions tested. The results shown are the average of three different experiments ± standard deviation. (B) IL-7 gene expression is strongly up-regulated in 3D cultured Hacat keratinocytes either alone or in the presence of fibroblasts. The differences between 3D and 2D either Hacat (*p<0.05) or co-cultures (**p<0.05) are significant. No differences were observed in the housekeeping gene expression in all the conditions tested. The results shown are the average of three different experiments ± standard deviation. (C) Time dependent up-regulation of the Dll-4 gene in three-dimensional Hacat keratinocytes/ fibroblasts co-cultures. A strong induction is observed during the first week and this high expression is maintained for about 10 days. The results shown are the average of three different experiments ± standard deviation and the differences between 2D and 3D within days 4-14 (* p < 0.01; ** p < 0.01; *** p <0.05; † p < 0.01; † † p < 0.01), are all statistically significant. (D) Dll-4 protein expression in 2D and 3D cultured Hacat keratinocytes. The western blot image shows different parts of one single gel. The average of Dll-4 level of expression normalized to actin from three different experiments ± standard deviation differs between the 2D and 3D environment and the difference is significant (p< 0.001).</p

TREC analysis shows that thymocytes were generated de novo.

<p>(A) TREC was amplified from DNA from cells generated in the matrices after 10 days of co-culture but not from DNA from cord blood separated precursors. RPS-29 housekeeping gene was amplified in both cases. TREC and RPS-29 were respectively identified as a band of 192 and 142 base pairs. (B) TREC/CD3⁺ ratios from cord blood T cells and thymocytes generated in the matrices. The latter cells show higher level of TREC expression per cell compared to T cells which were separated from cord blood. The results shown are the average of three different experiments± standard deviation and the difference is significant (p < 0.001).</p

Expansion and differentiation of CD34⁺ cells.

<p>(A) Correlation between the initial number of CD34⁺ cells seeded and the amount of mature cells generated at day 14^th. The results are the average ± standard derivation of three different experiments. (B) Progressive decline with time of CD34 expression among cord blood cellscultured in the matrix. The results are the average of three different experiments ± standard derivation. The differences between the 3^rd, 5^th and 14^th day and the seeded population are all significant (*p< 0.001; **p< 0.001; ***p< 0.001).</p

Generation of CD3+ thymocytes.

<p>(A) CD7^hiCD3^hi and CD7 ^dim CD3⁻ cells were detected at day 7. (B) By day 12 approximately 90% of all the cells generated were CD3⁺ thymocytes. (C) A matrix seeded with approximately 300 CD34⁺ cord blood derived progenitors generated about 2900 CD3⁺ cells after 14 days. At that time about 150 CD34⁺ progenitors were still present whereas no other cell types were detected. The image A is representative of three different experiments while images B and C show a single experiment.</p

Most of generated cells are mature thymocytes by day12.

<p>The presence of double positive CD4⁺CD8⁺ and either CD4⁺ or CD8⁺ single positive CD3⁺ thymocytes was evident by day 12 when only about 2% of total CD45⁺ cells still expressed CD34. The images are representative of three different experiments.</p