Light-activated Frizzled7 reveals a permissive role of non-canonical wnt signaling in mesendoderm cell migration

10.7554/eLife.42093.001Non-canonical Wnt signaling plays a central role for coordinated cell polarization and directed migration in metazoan development. While spatiotemporally restricted activation of non-canonical Wnt-signaling drives cell polarization in epithelial tissues, it remains unclear whether such instructive activity is also critical for directed mesenchymal cell migration. Here, we developed a light-activated version of the non-canonical Wnt receptor Frizzled 7 (Fz7) to analyze how restricted activation of non-canonical Wnt signaling affects directed anterior axial mesendoderm (prechordal plate, ppl) cell migration within the zebrafish gastrula. We found that Fz7 signaling is required for ppl cell protrusion formation and migration and that spatiotemporally restricted ectopic activation is capable of redirecting their migration. Finally, we show that uniform activation of Fz7 signaling in ppl cells fully rescues defective directed cell migration in fz7 mutant embryos. Together, our findings reveal that in contrast to the situation in epithelial cells, non-canonical Wnt signaling functions permissively rather than instructively in directed mesenchymal cell migration during gastrulation

アストロサイトにおける抗うつ薬による塩基性線維芽細胞増殖因子産生機構に関する薬理学的研究

内容の要旨 , 審査の要旨広島大学(Hiroshima University)博士(薬科学)Doctor of Philosophy in Medicinal Sciencedoctora

Peripheral Blood Single-Cell Genotyping and Phenotyping in Multiple Myeloma Reveals Shared Mutations across Multiple Hematopoietic Cell Lineages

Abstract Background: Multiple myeloma (MM) is a hematologic malignancy of terminally differentiated plasma cells. While most genomic studies of the disease have focused on characterizing isolated bone marrow plasma cells, few have addressed somatic mutations within the immune microenvironment. We previously demonstrated that somatic mutations are significantly more numerous in unsorted blood compared to bone marrow and that the number of mutations correlated with elevated markers of disease. We also reported that genes implicated in clonal hematopoiesis of indeterminate potential (CHIP) are commonly mutated in the blood from patients with MM. Aim: To integrate single-cell DNA mutation analysis and protein expression to map the clonal relationship between circulating plasma cells and non-plasma cells within the peripheral blood of patients with MM. Methods: Although bulk DNA sequencing can estimate clonal heterogeneity using analytic deconvolution techniques, it is unable to distinguish which mutations occur in the same clone(s) nor can it correlate genotype with immunophenotype from the same cell. To accurately describe the clonal architecture of the blood, we combined targeted single-cell DNA sequencing (scDNA-seq) coupled with protein expression to define mutations specific to cell phenotypes using the Tapestri Platform (Mission Bio). For this analysis, we developed a custom amplicon panel covering 22 frequently mutated genes in MM and/or CHIP to perform scDNA-seq and 4 antibodies targeting CD138, CD19, CD3, and CD33 for immunophenotyping or plasma cells, B cells, T cells, and myeloid cells respectively. Results: Peripheral blood mononuclear cells were isolated from 17 blood samples collected from 12 patients with MM. Three samples were collected at diagnosis before treatment, 8 were collected post-treatment during remission, and 6 were collected at relapse. Serial samples were obtained before and after autologous stem cell transplant from 4 patients. A total of 85,262 cells were sequenced uncovering 154 unique variants in 22 genes after quality filtering. There were 47 somatic variants, defined as those present in less than 40% of cells per sample. There were 52,136 total clones, defined as cells with identical genotypes, within this heterogenous population of cells. The most common non-synonymous somatic mutations were identified in genes SETD2, KMT2C, PPM1D. An immunophenotype was assigned to 82% of cells according to the protein with the most abundant number of reads. Dimensionality reduction of single-cell genotype using t-distributed stochastic neighbor embedding (t-SNE) effectively clustered cells according to sample and by patient in cases where there were multiple samples from the same individual. Patients with active myeloma and known circulating plasma cells detected by clinical flow cytometry were found to have the greatest proportion of CD138+ cells as well as the highest mutational burden. It was rare to detect the same clone in two separate samples collected from the same patient at different time points (median time be collection was 248 days) which may be due to under-sampling or clonal evolution. Surprisingly, all somatic variants detected in plasma cells, were also present in other cell types suggesting they were acquired from a common progenitor cell rather than after differentiation. Conclusions: scDNA-seq coupled with immunophenotyping of the peripheral blood of patients with MM reveals somatic variants are shared across multiple hematopoietic cell lineages including plasma cells indicating these mutational are acquired from a common progenitor. Disclosures Durruthy-Durruthy: Mission Bio Inc.: Current Employment. Arribas-Layton: Mission Bio, Inc.: Current Employment. Wang: Mission Bio, Inc: Current Employment

Distinct skeletal stem cell types orchestrate long bone skeletogenesis

Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research

a tool for real time bottlenose dolphin monitoring in the Portofino

Bottlenose dolphin (Tursiops truncatus) is one of the Mediterranean cetaceans listed in the Annex II of Habitat Directive. The main objective is the creation of a virtual corridor for monitoring and surveillance of the transient and resident bottlenose dolphins. Concrete conservation actions take place in the Portofino MPA (Italy). We show the implementation of an interference avoidance system capable to track the dolphins, to identify threats and to prevent collisions by diffusing real time warning messages to all categories involved. Two detection units are placed one kilometer off the coast of Portofino headland. Each unit is a particular type of marine buoy (elastic beacon) equipped with four hydrophones and an acquisition system which can record the typical \u201csocial communication whistles\u201d emitted by the dolphins and the sounds emitted by boat engines. Signals are then sent on shore, via wi-fi, and elaborated to get the real time position of dolphins and boats. Upon reception of the warnings the boats present in the area will be invited to follow a protocol of conduct supervised by the Coast Guard. This approach will improve the species protection, the sustainable coexistence of dolphins and anthropic activities and will promote responsible usage of the sea, especially in one of the most touristic Marine Protected Area in Mediterranean Sea. We illustrate the technical details of the automatic system for bottlenose dolphins conservation and results of first ten months of observation will be reported. This study is part of the Life+ Nature Project \u201cARION\u201d co-funded by the European Commission

Statistical evaluation of the background signal in acoustic data from ARION project

The ARION project aim is to protect dolphins and boats, while preserving human activities, mainly preventing collisions. To achieve this goal it is necessary to track both and to signal the presence of the dolphins to possibly any subject operating in the protected area. Two buoys with four hydrophones each, installed in Santa Margherita-Portofino marine protected area, collect data transmitted to a ground station. The average power of noise background is at the same time a parameter affecting resolution and an indicator of noise pollution. The equivalent sea state noise is calculated from this parameter and compared against Wentz curves

A Permanent Automated Real-Time Passive Acoustic Monitoring System for Bottlenose Dolphin Conservation in the Mediterranean Sea.

Within the framework of the EU Life+ project named LIFE09 NAT/IT/000190 ARION, a permanent automated real-time passive acoustic monitoring system for the improvement of the conservation status of the transient and resident population of bottlenose dolphin (Tursiops truncatus) has been implemented and installed in the Portofino Marine Protected Area (MPA), Ligurian Sea. The system is able to detect the simultaneous presence of dolphins and boats in the area and to give their position in real time. This information is used to prevent collisions by diffusing warning messages to all the categories involved (tourists, professional fishermen and so on). The system consists of two gps-synchronized acoustic units, based on a particular type of marine buoy (elastic beacon), deployed about 1 km off the Portofino headland. Each one is equipped with a four-hydrophone array and an onboard acquisition system which can record the typical social communication whistles emitted by the dolphins and the sound emitted by boat engines. Signals are pre-filtered, digitized and then broadcast to the ground station via wi-fi. The raw data are elaborated to get the direction of the acoustic target to each unit, and hence the position of dolphins and boats in real time by triangulation

Purification and functional characterization of novel human skeletal stem cell lineages

Human skeletal stem cells (hSSCs) hold tremendous therapeutic potential for developing new clinical strategies to effectively combat congenital and age-related musculoskeletal disorders. Unfortunately, refined methodologies for the proper isolation of bona fide hSSCs and the development of functional assays that accurately recapitulate their physiology within the skeleton have been lacking. Bone marrow-derived mesenchymal stromal cells (BMSCs), commonly used to describe the source of precursors for osteoblasts, chondrocytes, adipocytes and stroma, have held great promise as the basis of various approaches for cell therapy. However, the reproducibility and clinical efficacy of these attempts have been obscured by the heterogeneous nature of BMSCs due to their isolation by plastic adherence techniques. To address these limitations, our group has refined the purity of individual progenitor populations that are encompassed by BMSCs by identifying defined populations of bona fide hSSCs and their downstream progenitors that strictly give rise to skeletally restricted cell lineages. Here, we describe an advanced flow cytometric approach that utilizes an extensive panel of eight cell surface markers to define hSSCs; bone, cartilage and stromal progenitors; and more differentiated unipotent subtypes, including an osteogenic subset and three chondroprogenitors. We provide detailed instructions for the FACS-based isolation of hSSCs from various tissue sources, in vitro and in vivo skeletogenic functional assays, human xenograft mouse models and single-cell RNA sequencing analysis. This application of hSSC isolation can be performed by any researcher with basic skills in biology and flow cytometry within 1-2 days. The downstream functional assays can be performed within a range of 1-2 months