Functional Niche Research Articles

Oncostatin M regulates hematopoietic stem cell (HSC) niches in the bone marrow to restrict HSC mobilization.

We show that pro-inflammatory oncostatin M (OSM) is an important regulator of hematopoietic stem cell (HSC) niches in the bone marrow (BM). Treatment of healthy humans and mice with granulocyte colony-stimulating factor (G-CSF) dramatically increases OSM release in blood and BM. Using mice null for the OSM receptor (OSMR) gene, we demonstrate that OSM provides a negative feed-back acting as a brake on HSPC mobilization in response to clinically relevant mobilizing molecules G-CSF and CXCR4 antagonist. Likewise, injection of a recombinant OSM molecular trap made of OSMR complex extracellular domains enhances HSC mobilization in poor mobilizing C57BL/6 and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. Mechanistically, OSM attenuates HSC chemotactic response to CXCL12 and increases HSC homing to the BM signaling indirectly via BM endothelial and mesenchymal cells which are the only cells expressing OSMR in the BM. OSM up-regulates E-selectin expression on BM endothelial cells indirectly increasing HSC proliferation. RNA sequencing of HSCs from Osmr-/- and wild-type mice suggest that HSCs have altered cytoskeleton reorganization, energy usage and cycling in the absence of OSM signaling in niches. Therefore OSM is an important regulator of HSC niche function restraining HSC mobilization and anti-OSM therapy combined with current mobilizing regimens may improve HSPC mobilization for transplantation.

The divergent paths of pragmaticalization: The case of the Russian particles tipa and vrode

Based on a qualitative and quantitative examination of 2018–2019 online comments in a sizable student group on a popular social network, this paper peruses the various functions of two Russian polyfunctional particles derived from semantically similar sources, tipa and vrode. While the prepositional tipa ‘like’ and vrode ‘like’ are used synonymously as grammaticalized markers of (approximative) similarity and exemplification, the subjectification and pragmaticalization of the two items as discourse particles has followed divergent paths. Both particles pertain to the domains of evidentiality and epistemic modality and are related to certain ‘seemingliness’ and appearance which is contrasted with reality. However, they have acquired niche functions within these broad domains, activating and building upon different facets of the universal category of similitude. Vrode has been established as a marker of uncertainty (quasi-assertive) which implies a particular type of evidence – sensory perception, recall, or information received from other people. Another distinctive function of vrode is marking that an expectation dictated by the situation designated by the particle's host has not been met. Tipa, on the other hand, is used as an approximator, a quotative/reportative, a doubt or falsity marker, a marker of discourse-based inference, or an explanatory marker, as well as a depictive, or a focus device. This paper also reports that the particle tipa, which, unlike vrode, is stigmatized by normative grammars, is underrepresented in comments by women. This especially concerns tipa's most advanced uses – as a voice or stance shifter and a clarificatory-explanatory operator – which are conceptually further away from the original similative meaning.

Effects of intensive human management on the taxonomic and functional diversity of ground beetles in a planted forest landscape

The rapid expansion of planted forests harms native biodiversity. Few studies report the effects of replacing wetlands with planted forests on ground beetles. We analyze how the taxonomic and functional diversity of ground beetles are affected by intensive management of a planted forest landscape in the Lower Delta of the Parana River. We defined six habitat types (n = 3, N = 18): young and mature willow (YW, MW), young and mature poplar without cattle (YP, MP), young and mature silvopastoral poplar (YS, MS). Using pitfall traps (N = 1728), we recorded 35 species (1896 individuals). YW and MS reached the highest taxonomic diversity and richness. YW with more vertical heterogeneity showed higher species richness than MW. Hydrophilic species were more abundant in YW. Zoophagous species were more abundant in MS. YS, MS, and YW reached the highest functional evenness, which implies that a large part of the functional niches was used. Cattle dung and freshwater canals for livestock offer more resources for ground beetles. The planted tree species, stand age, and presence of cattle affects taxonomic and functional diversity of ground beetles. Willow and silvopastoral planted forests are the most suitable habitats for hosting wetland species. So, we recommend using willow species rather than poplar species when planted forests replace fluvial wetlands, increasing irrigation of poplar planted forests through ditches and canals, conserving or restoring different strata of understory to increase vertical heterogeneity, and maintaining the landscape heterogeneity. These management measures are essential to prevent the loss of wetland species and conserve ground beetle’s diversity.

Spatial and functional niche overlap between invasive Ligustrum lucidum and native woody species in an urban shrine forest in Japan

Spatial and functional niche overlap between invasive Ligustrum lucidum and native woody species in an urban shrine forest in Japan

Functional beta diversity of New Zealand fishes: Characterising morphological turnover along depth and latitude gradients, with derivation of functional bioregions

AbstractChanges in the functional structures of communities are rarely examined along multiple large‐scale environmental gradients. Here, we describe patterns in functional beta diversity for New Zealand marine fishes versus depth and latitude, including broad‐scale delineation of functional bioregions. We derived eight functional traits related to food acquisition and locomotion and calculated complementary indices of functional beta diversity for 144 species of marine ray‐finned fishes occurring along large‐scale depth (50–1200 m) and latitudinal gradients (29°–51°S) in the New Zealand Exclusive Economic Zone. We focused on a suite of morphological traits calculated directly from in situ Baited Remote Underwater Stereo‐Video (stereo‐BRUV) footage and museum specimens. We found that functional changes were primarily structured by depth followed by latitude, and that latitudinal functional turnover decreased with increasing depth. Functional turnover among cells increased with increasing depth distance, but this relationship plateaued for greater depth distances (>750 m). In contrast, functional turnover did not change significantly with increasing latitudinal distance at 700–1200 m depths. Shallow functional bioregions (50–100 m) were distinct at different latitudes, whereas deeper bioregions extended across broad latitudinal ranges. Fishes in shallow depths had a body shape conducive to efficient propulsion, while fishes in deeper depths were more elongated, enabling slow, energy‐efficient locomotion, and had large eyes to enhance vision. Environmental filtering may be a primary driver of broad‐scale patterns of functional beta diversity in the deep sea. Greater environmental homogeneity may lead to greater functional homogeneity across latitudinal gradients at deeper depths (700–1200 m). We suggest that communities living at depth may follow a ‘functional village hypothesis’, whereby similar key functional niches in fish communities may be maintained over large spatial scales.

Proliferative stem cells maintain quiescence of their niche by secreting the Activin inhibitor Follistatin

SummaryAging causes stem cell dysfunction as a result of extrinsic and intrinsic changes. Decreased function of the stem cell niche is an important contributor to this dysfunction. We use the Drosophila testis to investigate what factors maintain niche cells. The testis niche comprises quiescent “hub” cells and supports two mitotic stem cell pools: germline stem cells and somatic cyst stem cells (CySCs). We identify the cell-cycle-responsive Dp/E2f1 transcription factor as a crucial non-autonomous regulator required in CySCs to maintain hub cell quiescence. Dp/E2f1 inhibits local Activin ligands through production of the Activin antagonist Follistatin (Fs). Inactivation of Dp/E2f1 or Fs in CySCs or promoting Activin receptor signaling in hub cells causes transdifferentiation of hub cells into fully functional CySCs. This Activin-dependent communication between CySCs and hub regulates the physiological decay of the niche with age and demonstrates that hub cell quiescence results from signals from surrounding stem cells.

Cdc42 is required for male germline niche development in mice.

Spermatogonial stem cells (SSCs) are maintained in a special microenvironment called a niche. However, much is unknown about components that constitute the niche. Here, we report that Cdc42 is essential for germline niche development. Sertoli cell-specific Cdc42-deficient mice showed normal premeiotic spermatogenesis. However, germ cells gradually disappeared during haploid cell formation and few germ cells remained in the mature testes. Spermatogonial transplantation experiments revealed a significant loss of SSCs in Cdc42-deficient testes. Moreover, Cdc42 deficiency in Sertoli cells downregulated GDNF, a critical factor for SSC maintenance. Cdc42-deficient Sertoli cells also exhibited lower nuclear MAPK1/3 staining. Inhibition of MAP2K1 or depletion of Pea15a scaffold protein downregulated GDNF expression. A screen of transcription factors revealed that Cdc42-deficient Sertoli cells downregulate DMRT1 and SOX9, both of which are critical for Sertoli cell development. These results indicate that Cdc42 is essential for niche function via MAPK1/3-dependent GDNF secretion.

How do lizard niches conserve, diverge or converge? Further exploration of saurian evolutionary ecology

BackgroundEnvironmental conditions on Earth are repeated in non-random patterns that often coincide with species from different regions and time periods having consistent combinations of morphological, physiological and behavioral traits. Observation of repeated trait combinations among species confronting similar environmental conditions suggest that adaptive trait combinations are constrained by functional tradeoffs within or across niche dimensions. In an earlier study, we assembled a high-resolution database of functional traits for 134 lizard species to explore ecological diversification in relation to five fundamental niche dimensions. Here we expand and further examine multivariate relationships in that dataset to assess the relative influence of niche dimensions on the distribution of species in 6-dimensional niche space and how these may deviate from distributions generated from null models. We then analyzed a dataset with lower functional-trait resolution for 1023 lizard species that was compiled from our dataset and a published database, representing most of the extant families and environmental conditions occupied by lizards globally. Ordinations from multivariate analysis were compared with null models to assess how ecological and historical factors have resulted in the conservation, divergence or convergence of lizard niches.ResultsLizard species clustered within a functional niche volume influenced mostly by functional traits associated with diet, activity, and habitat/substrate. Consistent patterns of trait combinations within and among niche dimensions yielded 24 functional groups that occupied a total niche space significantly smaller than plausible spaces projected by null models. Null model tests indicated that several functional groups are strongly constrained by phylogeny, such as nocturnality in the Gekkota and the secondarily acquired sit-and-wait foraging strategy in Iguania. Most of the widely distributed and species-rich families contained multiple functional groups thereby contributing to high incidence of niche convergence.ConclusionsComparison of empirical patterns with those generated by null models suggests that ecological filters promote limited sets of trait combinations, especially where similar conditions occur, reflecting both niche convergence and conservatism. Widespread patterns of niche convergence following ancestral niche diversification support the idea that lizard niches are defined by trait-function relationships and interactions with environment that are, to some degree, predictable and independent of phylogeny.

Mechanical property gradients of taenioglossan radular teeth are associated with specific function and ecological niche in Paludomidae (Gastropoda: Mollusca)

Biological tissues may exhibit graded heterogeneities in structure and mechanical properties that are crucial to their function. One biological structure that shows variation in both structure and function is the molluscan radula: the organ comprises a chitinous membrane with embedded teeth and serves to process and gather food. The tooth morphologies had been well studied in the last decades, but the mechanical properties of the teeth are not known for the vast majority of molluscs. This knowledge gap restricts our understanding of how the radula is able to act effectively on a target surface whilst simultaneously resisting structural failure. Here we employed nanoindentation technique to measure mechanical properties (hardness and Young's modulus) on distinct localities of individual radular teeth from 24 species of African paludomid gastropods. These species have distinct ecological niches as they forage on algae on different feeding substrates. A gradual distribution of measured properties along the teeth was found in species foraging on solid or mixed feeding substrates, but soft substrate feeders exhibit teeth almost homogeneous in their biomechanical properties. The presence or absence of large-scale gradients in these taenioglossan teeth could directly be linked with their specific function and in general with the species ecology, whereas the radular tooth morphologies do not always and fully reflect ecology. Statement of significanceAfrican Lake Tanganyika is well known for harbouring endemic and morphologically distinct genera. Its paludomid gastropods form a flock of high interest because of its diversity. As they show distinct radular tooth morphologies hypotheses about potential trophic specializations had always been at hand. Here we evaluated the mechanical properties Young’s modulus and hardness of 9027 individual teeth from 24 species along the tooth by nanoindentation and related them with the gastropods’ specific feeding substrate. We find that hard substrate feeders have teeth that are hard at the tips but much less stiff at the base and thus heterogeneous with respect to material properties, whereas soft substrate feeders have teeth that are flexible and homogenous with respect to material properties.

A unifying framework for quantifying and comparing n‐dimensional hypervolumes

Abstract The quantification of Hutchinson's n‐dimensional hypervolume has enabled substantial progress in community ecology, species niche analysis and beyond. However, most existing methods do not support a partitioning of the different components of hypervolume. Such a partitioning is crucial to address the ‘curse of dimensionality’ in hypervolume measures and interpret the metrics on the original niche axes instead of principal components. Here, we propose the use of multivariate normal distributions for the comparison of niche hypervolumes and introduce this as the multivariate‐normal hypervolume (MVNH) framework (R package available on https://github.com/lvmuyang/MVNH). The framework provides parametric measures of the size and dissimilarity of niche hypervolumes, each of which can be partitioned into biologically interpretable components. Specifically, the determinant of the covariance matrix (i.e. the generalized variance) of a MVNH is a measure of total niche size, which can be partitioned into univariate niche variance components and a correlation component (a measure of dimensionality, i.e. the effective number of independent niche axes standardized by the number of dimensions). The Bhattacharyya distance (BD; a function of the geometric mean of two probability distributions) between two MVNHs is a measure of niche dissimilarity. The BD partitions total dissimilarity into the components of Mahalanobis distance (standardized Euclidean distance with correlated variables) between hypervolume centroids and the determinant ratio which measures hypervolume size difference. The Mahalanobis distance and determinant ratio can be further partitioned into univariate divergences and a correlation component. We use empirical examples of community‐ and species‐level analysis to demonstrate the new insights provided by these metrics. We show that the newly proposed framework enables us to quantify the relative contributions of different hypervolume components and to connect these analyses to the ecological drivers of functional diversity and environmental niche variation. Our approach overcomes several operational and computational limitations of popular nonparametric methods and provides a partitioning framework that has wide implications for understanding functional diversity, niche evolution, niche shifts and expansion during biotic invasions, etc.

Niche Characterization of Shrub Functional Groups along an Atlantic-Mediterranean Gradient

The identification of the factors controlling the understory species distribution and abundance is essential to understand the ecology and dynamics of natural forests and their management response. We assess the relationships between environmental gradients and shrub functional groups distribution patterns and niche characteristics in a transitional area between the Eurosiberian and Mediterranean biogeographic regions in Northern Spain. Here, 772 plots from the 3rd Spanish National Forest Inventory were used. Shrub functional groups respond to the same complex environmental gradients as trees, i.e., the north-south climatic gradient and a slope gradient. Unimodal response curves of shrub functional groups and families dominate along both gradients, providing evidence of successful functional turnover. Similar to tree species, the niche location of functionally related shrubs is close. Functional groups occupying environments with sharp contrast or transitional environments have the broadest niches, whereas those specialized functional groups occupying localized habitats showed the narrowest niches. The knowledge of shrub species distributions and niche characteristics along complex environmental gradients will improve our ability to discuss potential conservation management goals or threats due to land-use changes and future climate change.

Diazotrophic Cyanobacteria are Associated With a Low Nitrate Resupply to Surface Waters in Lake Tanganyika

In Lake Tanganyika, blooms of nitrogen-fixing (diazotrophic) cyanobacteria emerge, when the upper water column re-stratifies after a period of upwelling and convective mixing. During this seasonal transition, diazotrophic cyanobacteria exploit the abundant phosphate and fix nitrogen after other phytoplankton taxa have consumed the available nitrate. However, it remains less clear, which mechanisms favour diazotrophic cyanobacteria under more heavily stratified conditions with lower levels of excess phosphate and persistent nitrate-depletion. Here, we collected profiles of physicochemical parameters, nutrients and photo-pigments, as well as the medium- to large-sized phytoplankton community during two lake-wide cruises to elucidate to what extent the abundance of diazotrophic cyanobacteria in Lake Tanganyika may be controlled by the nitrate resupply through the thermocline into the euphotic zone. At stations where nitrate was depleted, but phosphate remained available near the surface, high densities of diazotrophic cyanobacteria were associated with a low nitrate supply to surface waters. Our data provide first support for two conceptual scenarios, where the relative position of the thermocline and the euphotic depth may create a functional niche for diazotrophic cyanobacteria: when the upward transport of nitrate into the euphotic zone is reduced by a subjacent thermocline, diazotrophic cyanobacteria, comprising Dolichospermum and Anabaenopsis, are key players in the medium-to large-sized phytoplankton community. By contrast, a thermocline located within the euphotic zone allows for a rapid vertical transport of nitrate for a thriving nitrate-assimilating phytoplankton community that evidently outcompetes diazotrophic cyanobacteria. This study highlights that, under nitrogen-depleted conditions, diazotrophic cyanobacteria can also grow in response to a reduced nutrient resupply to the productive surface waters.

What is different about different net-zero carbon electricity systems?

In deeply decarbonized electricity systems with significant shares of variable renewable energy, the additional availability of at least one firm electricity generating technology can overcome reliability challenges and substantially reduce electricity costs. Firm resources can operate at any time of the year and for as long as needed to maintain electricity system reliability. Low- and zero-carbon firm technologies include flexible resources with high variable and low capital costs, such as biogas or hydrogen combustion, capital-intensive resources with low or zero variable cost, including nuclear and geothermal, as well as intermediate resources such as natural gas plants with carbon capture and sequestration (CCS). This paper explains the distinct roles of nuclear, CCS, and combustion of zero-carbon fuels in decarbonized electricity systems as examples of each class of firm resources. We analyze and compare results from three long-term electricity system capacity expansion models for California and the U.S. Western Interconnection, demonstrating robustness of our conclusions to different model assumptions and domains. Individually, each firm technology delivers substantial cost reductions relative to portfolios restricted to wind, solar, and energy storage alone. Additionally, because each technology occupies a distinctive functional niche in the electricity system, having all of these technologies available optimizes the utilization rate of each resource and reduces system costs by up to 10% relative to cases with just one class of firm resource. The analysis highlights the benefits of an expansive range of technology options to meet emissions reductions goals for the power sector while maintaining operational reliability and affordability.

Niches that regulate stem cells and hematopoiesis in adult bone marrow.

In mammals, hematopoietic stem cells (HSCs) engage in hematopoiesis throughout adult life within the bone marrow, where they produce the mature cells necessary to maintain blood cell counts and immune function. In the bone marrow and spleen, HSCs are sustained in perivascular niches (microenvironments) associated with sinusoidal blood vessels-specialized veins found only in hematopoietic tissues. Endothelial cells and perivascular leptin receptor+ stromal cells produce the known factors required to maintain HSCs and many restricted progenitors in the bone marrow. Various other cells synthesize factors that maintain other restricted progenitors or modulate HSC or niche function. Recent studies identified new markers that resolve some of the heterogeneity among stromal cells and refine the localization of restricted progenitor niches. Other recent studies identified ways in which niches regulate HSC function and hematopoiesis beyond growth factors. We summarize the current understanding of hematopoietic niches, review recent progress, and identify important unresolved questions.

#45: Phenotypic heterogeneity among isolates of Candida albicans from specific anatomical niches in VLBW premature infants

Abstract Background Disseminated candidiasis is associated with high morbidity and mortality among very low birth weight (VLBW) infants. Research about C. albicans traits which lead to colonization versus invasive disease is lacking. Ability to grow in a filamentous form is a known virulence trait of C. albicans. Nevertheless, significant heterogeneity in filamentous growth in C. albicans clinical isolates has been documented (Hirakawa et. al). However, the clinical isolates investigated were obtained from varied tissue niches from hosts with differing risk factors, which could account for the variation in filamentation. A collection of C. albicans isolates prospectively collected from VLBW infants and their mothers demonstrated vertical transmission of C. albicans from mother to infant (Bliss et. al). We used the infants’ isolates from this collection to investigate filamentation amongst isolates obtained from the same tissue niche and host type. Methods The standard C. albicans reference strain in the field, SC5314 was chosen for a positive control as it demonstrates strong filamentation and high virulence. A strain lacking Efg1, a regulatory transcription factor that promotes filamentation, produces hypofilamentous colonies and was used as the negative control. Reference strains and the clinical isolates were grown on two types of stimulation media at 37 C for 7 days after which filamentation was scored. Results Filamentation development varied widely among isolates. Of the strains which were acquired through vertical transmission, 5 of 11 strains showed strong filamentation, 5 of 11 had an intermediate phenotype, and 1 did not display any filamentation. Three strains were acquired through horizontal transmission (RO10, RO54, and RO56). Of these, one showed strong filamentation whereas the other two failed to form filaments. None of the neonatal isolates showed colony wrinkling, a prominent phenotype in the reference strain SC5314. Strain RO61, an isolate associated with candidemia showed strong filamentation. In contrast, strain RO39, which was associated with dermatitis, was non-filamentous. Conclusions We observed phenotypic heterogeneity with respect to filament development among clinical isolates obtained from the same niche (rectal) in similar hosts (VLBW premature infants). This is consistent with the variation seen in Hirakawa et al and indicates that phenotypic variation is not a function of host risk factors nor clinical niche. Filamentation was not required for vertical transmission. No conclusions can be drawn based on this observation with only 2 strains, but it is interesting to note that the isolate which caused candidemia and death of the patient was highly filamentous whereas the isolate that caused dermatitis was not. Further investigation of these strains, including genomic sequencing and additional phenotypic evaluations, will explore the underlying factors leading to transmission and virulence of C. albicans in VLBW premature infants.

Root stem cell niche networks: it's complexed! Insights from Arabidopsis.

The presence of two meristematic cell populations in the root and shoot apex allows plants to grow indefinitely. Due to its simple and predictable tissue organization, the Arabidopsis root apical meristem remains an ideal model to study mechanisms such as stem cell specification, asymmetric cell division, and differentiation in plants. The root stem cell niche consists of a quiescent organizing centre surrounded by mitotically active stem cells, which originate all root tissues. The transcription factors PLETHORA, SCARECROW, and WOX5 form signalling hubs that integrate multiple inputs from an increasing number of proteins implicated in the regulation of stem cell niche function. Recently, locally produced auxin was added to the list of important mobile factors in the stem cell niche. In addition, protein–protein interaction data elegantly demonstrate how parallel pathways can meet in a common objective. Here we discuss how multiple networks converge to specify and maintain the root stem cell niche.

Loss of ASXL1 in the Bone Marrow Niche Dysregulates Hematopoietic Stem Cell Fates through RNA Polymerase II Complex

Introduction: Somatic or de novo mutations of Additional Sex Combs-like 1 (ASXL1) frequently occur in patients with myeloid malignancies or Bohring-Opitz syndrome, respectively. Conditional deletion of Asxl1 in hematopoietic system (Asxl1Δ/Δ, Abdel-Wahab et al. J Exp Med 2013) or global loss of Asxl1 (Asxl1-/-, Wang et al. Blood 2014) resulted in myeloid malignancies in mice. Interestingly, hematopoietic disease phenotypes are severer in Asxl1-/- mice than that in Asxl1Δ/Δ mice. The difference in disease severity between the two different mouse models led us to hypothesize that ASXL1 plays an important role in the function of marrow niche for normal hematopoiesis and deletion of Asxl1 in the niche contributes to the hematopoietic defect in vivo.Methods: In the current study, we performed a serial hematopoietic phenotypic analyses in OsxCre;Asxl1fl/fl mice, in which Asxl1 is only deleted in the mesenchymal stromal cells (MSCs) and preosteoblasts. Bone marrow (BM) transplantation was performed to identify the biological effects of ASXL1 loss in the niche on hematopoietic stem cell (HSC) functions. Convergent analyses of RNA-seq and ChIP-seq data were performed to survey ASXL1 target genes that are critical for HSC maintenance.Results:OsxCre;Asxl1fl/fl mice had a reduced LT-HSC pool and skewed cell differentiation favoring granulocytic/monocytic lineages in the BM by flow cytometric analyses. Co-culture of MSC with Lin-cKit+ (LK) cells revealed that Asxl1-/- MSCs induced a preferential myeloid differentiation of LK cells compared with WT MSCs. BM transplantation assay further confirmed that deletion of Asxl1 in MSCs/preosteoblasts resulted in a reduced HSC pool and a preferential myeloid lineage increment. Gene set enrichment analysis of RNA-seq data showed that downregulated genes in Asxl1-/- MSCs were enriched in cell cycle, stem cell division/proliferation, and cell surface receptor signaling pathway. Gene ontology analysis revealed that some of the differentially expressed genes were associated with cell cycle, cell division/proliferation, extracellular matrix organization, and cell adhesion. Protein mass spectrometry analysis and immunoprecipitation demonstrated a novel interaction of ASXL1 with multiple core subunits of RNA polymerase II (RNAPII) complex. Gel filtration chromatography assay verified the interaction between ASXL1 and RNAPII. ChIP-seq analysis using ASXL1 and RNAPII antibodies showed a high degree of the overlap peaks between ASXL1 and RNAPII bound genes. Convergent analyses of RNA-seq and ChIP-seq data revealed that loss of Asxl1 deregulated RNAPII transcriptional function and altered the expression of genes critical for HSC maintenance, such as Cxcl1, Cxcl2, and Vcam1 . Importantly, ASXL1 expression decreased markedly in MSCs of patients with chronic myelomonocytic leukemia (CMML-MSCs) compared with that in healthy donors. Consistently, co-culture of CMML-MSCs with cord blood CD34+ cells revealed that CMML-MSCs had a reduced hematopoietic supportive activity and induced a myeloid differentiation bias of CD34+ cells.Conclusion: Our in vivo studies demonstrate that Asxl1 in MSCs/preosteoblasts is required for normal hematopoiesis through regulating RNAPII transcriptional function. This study provides a mechanistic insight into the function of ASXL1 in the niche to maintain normal hematopoiesis; and ASXL1 alteration in, at least, a subset of the niche cells induces myeloid differentiation bias, thus, contributes the progression of myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Macrophage Involvement in the Response of Acute Myeloid Leukaemia to Chemotherapy

Acute myeloid leukemia (AML) is a clonally heterogeneous neoplasm with poor survival rates. Although chemotherapy often leads to clinical remission, the relapse rate of AML is very high and usually fatal. Chemotherapy is usually effective at debulking AML blasts however a small population capable of re-initiating the leukemia (LIC) survive ultimately causing relapse. Mouse models of AML suggest that LIC are protected from chemotherapy by a protective niche similar to hematopoietic stem cell (HSC) niches. As 1) bone marrow (BM) macrophages are key regulators of HSC niche function and 2) tumor-associated macrophages promote chemotherapy resistance in several models of solid tumors (e.g. breast, lung & ovarian) we investigated a potential role of BM non-malignant macrophages in promoting chemoresistance of AML blasts.Using a mouse model of AML driven by the MLL-AF9 oncogene in which mouse HSC are transduced with MSC-MLL/AF9-ires-GFP and transplanted into syngeneic recipients, we examined how AML progression alters the phenotype of non-malignant GFP-negative BM macrophages by flow cytometry. 18 days post-transplant, GFP+ leukemia burden in the BM was in the 40-60% range. In non-malignant GFPneg BM macrophages, cell surface expression of MHC class II, CD80, L-selectin and neuropilin-1 was significantly decreased suggesting that AML blasts alter the function of BM macrophages.To determine whether BM macrophages could have any role in AML response to chemotherapy, we first determine whether non-malignant BM macrophages were depleted in vivo by treatment with cytarabine (5 days, 100mg/kg) and doxorubicin (3 days, 1mg/kg). Two days after cytarabine+doxoR treatment, most CD11b+ F4/80- Ly6G+ granulocytes and CD11b+ F4/80+ CD169- monocytes were ablated from the BM with 0.1% granulocytes and 1.0 % monocytes surviving after treatment. In contrast CD11b- F4/80+ CD169+ VCAM1+ macrophages were the most resilient myeloid cells in the BM with 9.5% still present 2 days after chemotherapy.To further investigate a possible role of these chemotherapy-resilient CD169+ macrophages in promoting response of AML blasts to chemotherapy, we transplanted MLL-AF9 leukemia cells generated with wild-type HSC into CD169DTR/WT mouse recipients in which one CD169 allele is knocked-in with human diphtheria toxin receptor (DTR). This enables specific depletion of CD169+ macrophages in vivo by injecting diphtheria toxin (DT). In this model, DT treatment targets host CD169+ macrophages but not the AML blasts as they do not contain the DTR knock-in. In the absence of chemotherapy, depletion of recipient CD169+ macrophages did not alter leukemia burden in the BM, spleen or blood 48 hours post depletion and did not alter mouse survival (median survival 29-30 days in both CD169-DTR and WT recipients treated with DT or saline).However when cytarabine + doxoR were administered 48 hours after DT administration, median survival of CD169-DTR recipients was significantly increased to 39.5 days compared to wild-type recipients 35 days, p<0.001. Furthermore leukemia burden in both the blood and spleen was significantly reduced.To determine whether CD169 could by itself mediate lower response of AML to chemotherapy, we transplanted MLL-AF9 cells generated from WT HSC into CD169DTR/DTR mice which are knocked-out for both CD169 alleles. Deletion of both CD169 alleles in recipient mice did not change mouse survival in response to chemotherapy in the absence of DT. Therefore absence of CD169 is not sufficient to prolong mouse survival in response to chemotherapy.In conclusion, non-malignant CD169+ macrophages may protect MLL-AF9 leukemia blasts from the cytotoxic effects of cytarabine and doxorubicin. The mechanisms of this chemoprotection remain to be determined but do not involve a direct effect of CD169 on AML blasts. DisclosuresNo relevant conflicts of interest to declare.

CXCL12-Expressing Bone Marrow Niches Differentially Regulate CML Compared to Normal Stem Cells

CXCL12 plays a major role in hematopoietic stem cells (HSC) localization to regulatory niches in the bone marrow microenvironment (BMM). Previous studies indicate that CXCL12 deletion from Prx+ mesenchymal stem cells (MSC) reduces murine HSC numbers, and from Tek+ endothelial cells (EC) modestly reduces their repopulating activity. In contrast, CXCL12 deletion from Ocn+ osteoblasts or Osx+ osteoprogenitors does not affect HSC function (Greenbaum et al. Nature. 2013 Mar 14;495(7440):227-30; Ding et al. Nature. 2013 Mar 14;495(7440):231-5.). These studies identified MSC and endothelial cells as important HSC niche components. However, niche cells for CML leukemic stem cells (LSC) remain poorly characterized. To evaluate the contribution of CXCL12-expressing populations to LSC regulation, we transplanted normal and CML BM cells (CD45.1/2+; 2*106/mouse) into CXCL12f/f (loxP sites flanking exon 2) mice crossed with Tek-Cre and Prx-Cre mice ( reported in ASH 2016, Blood, Abstract: 26) . We showed that CXCL12 deletion from Prx+ MSC resulted in reduction in normal HSC (LSK Flt3- CD150+ CD48- cells) numbers and function compared to controls, but did not observe significant reduction of HSC with CXCL12 deletion from Tek+ EC. Interestingly, transplantation of CML cells into mice with CXCL12 deleted from Prx+ MSC led to reduced survival compared to control mice associated with increased WBC counts, BM cellularity, LSC numbers, and secondary repopulation capacity. In contrast, CXCL12 deletion from Tek+ cells led to enhanced survival, associated with reduced BM cellularity and reduced LSC numbers. In further studies, we transplanted mixtures of equal numbers of normal and CML cells into Prx-Cre and Tek-Cre mice, and found a marked growth advantage of LSC compared with HSC in Prx-Cre mice, and a growth advantage of HSC over LSC in Tek-Cre knockout mice. We also show that both HSC and LSC demonstrate increased cell cycling via EdU labeling. Consistent with this, we found that Nilotinib treatment leads to significantly greater reduction in BM cellularity, LSC numbers and secondary repopulation potential in Prx-Cre mice, compared to Tek-Cre or WT mice. To evaluate the effects of CML on CXCL12-expressing BMM cells, we crossed CXCL12GFP (GFP reporter knocked into the CXCL12 locus) and SCL-tTA-BCR-ABL mice to generate CXCL12GFP-BCR-ABL mice. CML development lead to significantly reduced numbers of GFP+ BM stromal cells (CD45-Ter119-31-) but increased GFP+ endothelial cells (CD45-Ter119-CD31+), and within the stromal population, the number of GFP+ MSC (PDGFRα+Sca-1+) were decreased compared to WT mice. To further understand niche mediated interactions that regulate LSC fate, we studied gene expression profiles of LSC from mice following Cre-mediated targeting of CXCL12, compared to LSC from Cre-negative mice. Our preliminary analysis indicates significant enrichment of genes associated with cell cycle progression and Myc activation, and negative enrichment of genes related to TGF-b, STAT3, and Polycomb Repressive Complex 2 (PRC2) activity in LSC from Prx1+Cre CXCL12f/f mice. Reduced TGF-b, STAT3 and PRC2/EZH2 activity represent potential mechanisms for regulation of CML LSC maintenance and/or TKI resistance, and may potentially contribute to Prx1+ MSC-mediated LSC regulation. We conclude that CXCL12+ BM MSC inhibit CML development, maintain LSC quiescence, and enhance TKI resistance, while CXCL12+ EC enhance LSC maintenance and disease development. Leukemia associated reduction in CXCL12+MSC and increase in CXCL12+EC may enhance CML development. Collectively, these results reveal important and distinct niche functions for CXCL12 expressing BM MSC and EC for CML LSC compared with normal HSC. DisclosuresNo relevant conflicts of interest to declare.

The JAK2V617F-Bearing Megakaryocytes Promote Hematopoietic Stem/Progenitor Cell Expansion through the Thrombopoietin/MPL Signaling

IntroductionThe myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The acquired mutation JAK2V617F plays a central role in these disorders. The mechanisms responsible for MPN HSPC expansion are not fully understood. One hallmark of MPNs is megakaryocytic (MK) hyperplasia. Very recent evidence has implicated MKs in regulating HSPC activity. Since thrombopoietin (TPO) acting through its receptor MPL are key regulators of both megakaryopoiesis and HSPC activity, we hypothesize that JAK2V617F-bearing MKs promote MPN stem cell function via altered TPO/MPL signaling.MethodsJAK2V617F Flip-Flop (FF1) mice and Pf4-Cre mice were crossed to generate a MK cell lineage-specific human JAK2V617F knock-in mouse strain (Pf4/FF1). TPO or MPL was ablated in Pf4/FF1 by crossing Pf4/FF1 mice with TPO knock-out mice (TPOnull) or MPL knockout mice (MPLnull) to generate TPOnullPf4+FF1+ mice and MPLnullPf4+FF1+ mice. Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline.ResultsPreviously, we have demonstrated that JAK2V617F-bearing MKs cause a murine myeloproliferative syndrome with HSPC expansion in a Pf4/FF1 murine model. Here, we studied the effects of the JAK2V617F-bearing MK niche on MPN stem cell function. First, we isolated LineagenegcKit+ (Lin-cKit+) HSPCs from 28wk old Pf4/FF1 mice and their age-matched littermate controls (Pf4-Cre) (n=4 in each group) and analyzed their cell cycle status using propidium iodide staining. We found that the percentage of Lin-cKit+ cells in the G0/G1 phase was moderately increased in the Pf4/FF1 mice compared to control mice (63% vs 56%, P=0.012), suggesting that HSPCs in the JAK2V617F-bearing MK niche are more quiescent than in the WT MK niche. (Figure 1)Next, we performed a competitive repopulation assay in which both CD45.2 marrow cells from Pf4/FF1 mice or control mice were injected intravenously together with competitor CD45.1 WT marrow cells into lethally irradiated (950 cGy) CD45.1 recipients. During 16 weeks follow up post transplantation, recipients of Pf4/FF1 marrow cells displayed a higher peripheral blood donor (CD45.2) chimerism than the control mice. Marrow cell colony assays demonstrated a moderate increase of CD45.2+ hematopoietic progenitors in the Pf4/FF1 marrow recipients compared to control mice (1.5-fold, p = 0.0025). Marrow cell flow cytometry analysis revealed that, although there was no significant difference in the total CD41+ MK cell numbers between the two groups, CD45.2+ MKs were significantly increased (3.3-fold, P=0.012) while CD45.1+ MKs were significantly decreased (3.4-fold, P=0.024) in the Pf4/FF1 marrow recipient compared to the controls. Similarly, although the total numbers of marrow CD45+EPCR+CD48-CD150+ (E-SLAM) cells, which is a highly purified long-term repopulating HSPC population, were not changed between the two groups, CD45.2+ E-SLAM cells were significantly increased (3.5-fold, P=0.012) while CD45.1+ E-SLAM cells were significantly decreased (5.4-fold, P=0.024) in the Pf4/FF1 marrow recipient compared to the controls. Therefore, the JAK2V617F-bearing MK niche promoted the expansion of JAK2V617F HSPCs, probably at the expense of suppressing WT hematopoiesis. No significant difference in blood cell count, spleen weight, or total femoral cell count was observed between the Pf4/FF1 marrow recipients and control marrow recipients. (Figure 2)To determine the role(s) of TPO/MPL signaling in the development of JAK2V617F MK-induced myeloproliferation, we ablated TPO or MPL in the Pf4/FF1 mice. At 28 weeks of age, in contrast to the Pf4/FF1 mice which developed thrombocytosis, increased numbers of hematopoietic progenitors, and a substantial expansion of MKs and HSPCs compared to age-matched WT controls, TPOnullPf4+FF1+ mice and MPLnullPf4+FF1+ mice did not display any of these features. (Figure 3) These data suggest that TPO and MPL are required for the development of JAK2V617F MK-induced myeloproliferation.ConclusionsOur study establishes that JAK2V617F-bearing MKs form an important part of the MPN HSPC niche and TPO/MPL signaling is critical for the MK niche function. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.