IntroductionThe majority of hematopoietic progenitor cells (HPCs) reside in the bone marrow surrounded by a complex, highly organized microenvironment. Under normal conditions, a small number of HPCs are released into the peripheral blood. Agents with distinct cellular targets and biologic activities can induce the mobilization of HPCs into blood, including hematopoietic growth factors, chemotherapeutic agents, and chemokines. 1,2 Recently, mobilized peripheral blood HPCs have become the principal cellular source for reconstitution of the hematopoietic system following myeloablative therapy. Currently, granulocyte colony-stimulating factor (G-CSF) is the most widely used agent to induce HPC mobilization due to its potency, predictability, and safety. 3 However, the mechanisms responsible for G-CSF-induced HPC mobilization have not been defined.We previously showed that G-CSF receptor (G-CSFR) expression on HPCs is not required for their mobilization by G-CSF, suggesting that G-CSF induces HPC mobilization indirectly through the generation of trans-acting signals. 4 The nature of the transacting signals that mediate G-CSF-induced HPC mobilization is unknown; however, accumulating evidence suggests that interaction of CXCL12 (stromal-derived factor 1 [SDF-1]) with its cognate receptor, CXCR4 (CXC motif, receptor 4), may play an important role in regulating G-CSF-induced HPC mobilization. CXCL12 is a CXC chemokine constitutively produced in the bone marrow by stromal cells. 5 Studies of CXCL12-or CXCR4-deficient mice have established that these genes are necessary for the normal migration of HPCs from the fetal liver to the bone marrow and in the efficient retention of myeloid precursors in the adult bone marrow. 6,7 Moreover, treatment with AMD-3100, a specific antagonist of CXCR4, induces rapid and robust HPC mobilization in both humans and mice. 8,9 Finally, we and others showed that CXCL12 protein expression in the bone marrow is significantly decreased following G-CSF treatment. [10][11][12] Collectively, these data suggest a model in which disruption of CXCL12/CXCR4 signaling is a key step in G-CSF-induced HPC mobilization.The mechanisms mediating the G-CSF-induced decrease in CXCL12 protein expression in the bone marrow have not been For personal use only. on May 12, 2018. by guest www.bloodjournal.org From defined. Previous reports suggested that neutrophil elastase (NE) and cathepsin G (CG) might regulate CXCL12 protein expression in the bone marrow through proteolytic cleavage of CXCL12. 10,11 However, mice genetically lacking NE and CG display normal G-CSF-induced HPC mobilization, and the expected decrease in bone marrow CXCL12 protein was observed. 13 Thus, the G-CSFinduced decrease in CXCL12 protein expression in the bone marrow does not require these proteases. It is possible that other proteases can compensate for the loss of NE and CG. Alternatively, nonproteolytic mechanisms may regulate CXCL12 expression in the bone marrow during G-CSF-induced HPC mobilization.In this study, we characterize G-CSF...

Neutrophils are released from the bone marrow in a regulated fashion to maintain homeostatic levels in the blood and to respond to physiological stresses, including infection. We show that under basal conditions granulocyte colony-stimulating factor (G-CSF) is an essential regulator of neutrophil release from the bone marrow. Nonredundant signals generated by the membrane-proximal 87 amino acids of the G-CSF receptor (G-CSFR) are sufficient to mediate this response. Surprisingly, G-CSFR expression on neutrophils is neither necessary nor sufficient for their mobilization from the bone marrow, suggesting that G-CSF induces neutrophil mobilization indirectly through the generation of trans-acting signals. Evidence is provided suggesting that downregulation of stromal cell-derived factor 1 expression in the bone marrow may represent such a signal.

Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.

SUMMARY Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin+ osteoblasts (N-cad+OBs that enrich osteoprogenitors) in the niche. We further found that N-cad+OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca2+-NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche.

Neural crest (NC) cell migration is crucial to the formation of peripheral tissues during vertebrate development. However, how NC cells respond to different microenvironments to maintain persistence of direction and cohesion in multicellular streams remains unclear. To address this, we profiled eight subregions of a typical cranial NC cell migratory stream. Hierarchical clustering showed significant differences in the expression profiles of the lead three subregions compared with newly emerged cells. Multiplexed imaging of mRNA expression using fluorescent hybridization chain reaction (HCR) quantitatively confirmed the expression profiles of lead cells. Computational modeling predicted that a small fraction of lead cells that detect directional information is optimal for successful stream migration. Single-cell profiling then revealed a unique molecular signature that is consistent and stable over time in a subset of lead cells within the most advanced portion of the migratory front, which we term trailblazers. Model simulations that forced a lead cell behavior in the trailing subpopulation predicted cell bunching near the migratory domain entrance. Misexpression of the trailblazer molecular signature by perturbation of two upstream transcription factors agreed with the in silico prediction and showed alterations to NC cell migration distance and stream shape. These data are the first to characterize the molecular diversity within an NC cell migratory stream and offer insights into how molecular patterns are transduced into cell behaviors.

Hematopoiesis is a tightly controlled process maintained by a small pool of hematopoietic stem cells (HSCs). Here, we demonstrate that the LT-HSC, MPP, premegakaryocytic/erythroid, Pre CFU-E, Pre GM, MkP, and granulocyte-macrophage compartments were all significantly reduced in E2A-deficient bone marrow. Despite a severe depletion of erythroid progenitors, the erythrocyte and megakaryocyte compartments were equivalent in E2A-deficient bone marrow as compared with wild-type mice. E2A-deficient HSCs also failed to efficiently maintain the HSC pool on serial transplantation, and we demonstrate that the E2A proteins regulate cell cycle progression of HSCs by regulating the expression of p21 Cip1 , p27 Kip1 , and the thrombopoietin receptor, known regulators of HSC self-renewal activity. Based on these observations, we propose that the E2A proteins promote the developmental progression of the entire spectrum of early hematopoietic progenitors and to suppress an erythroid specific program of gene expression in alternative cell lineages. Last, the data mechanistically link E2A, cell cycle regulators, and the maintenance of the HSC pool in a common pathway.E47 ͉ lymphoid/myeloid versus erythroid/megakaryocyte development ͉ self-renewal

Embryonic neural crest cells travel in discrete streams to precise locations throughout the head and body. We previously showed that cranial neural crest cells respond chemotactically to vascular endothelial growth factor (VEGF) and that cells within the migratory front have distinct behaviors and gene expression. We proposed a cell-induced gradient model in which lead neural crest cells read out directional information from a chemoattractant profile and instruct trailers to follow. In this study, we show that migrating chick neural crest cells do not display distinct lead and trailer gene expression profiles in culture. However, exposure to VEGF in vitro results in the upregulation of a small subset of genes associated with an in vivo lead cell signature. Timed addition and removal of VEGF in culture reveals the changes in neural crest cell gene expression are rapid. A computational model incorporating an integrate-and-switch mechanism between cellular phenotypes predicts migration efficiency is influenced by the timescale of cell behavior switching. To test the model hypothesis that neural crest cellular phenotypes respond to changes in the VEGF chemoattractant profile, we presented ectopic sources of VEGF to the trailer neural crest cell subpopulation and show diverted cell trajectories and stream alterations consistent with model predictions. Gene profiling of trailer cells that diverted and encountered VEGF revealed upregulation of a subset of 'lead' genes. Injection of neuropilin1 (Np1)-Fc into the trailer subpopulation or electroporation of VEGF morpholino to reduce VEGF signaling failed to alter trailer neural crest cell trajectories, suggesting trailers do not require VEGF to maintain coordinated migration. These results indicate that VEGF is one of the signals that establishes lead cell identity and its chemoattractant profile is critical to neural crest cell migration.

Neural crest cells migrate throughout the embryo, but how cells move in a directed and collective manner has remained unclear. Here, we perform the first single-cell transcriptome analysis of cranial neural crest cell migration at three progressive stages in chick and identify and establish hierarchical relationships between cell position and time-specific transcriptional signatures. We determine a novel transcriptional signature of the most invasive neural crest Trailblazer cells that is consistent during migration and enriched for approximately 900 genes. Knockdown of several Trailblazer genes shows significant but modest changes to total distance migrated. However, in vivo expression analysis by RNAscope and immunohistochemistry reveals some salt and pepper patterns that include strong individual Trailblazer gene expression in cells within other subregions of the migratory stream. These data provide new insights into the molecular diversity and dynamics within a neural crest cell migratory stream that underlie complex directed and collective cell behaviors.