Embryonic hematopoietic cells develop in the fetal liver (FL), surrounded by diverse non-hematopoietic stromal cells. However, the spatial organization and cytokine production patterns of the stroma during FL development remain poorly understood. Here, we characterized and mapped the hematopoietic and stromal cell populations at early (E12.5-14.5) FL stages, revealing that while hepatoblasts were the primary source of hematopoietic growth factors, other stromal cells-including mesenchymal, mesothelial, and endothelial cells-also contributed to this signaling network. Using a dedicated image analysis pipeline, we quantified cell distances to tissue structures and defined neighbor relationships, uncovering that different hematopoietic progenitors exhibit distinct preferences for neighboring stromal cells and show developmental changes in spatial distribution. Notably, our data suggest that the sub-mesothelium region plays a prominent role in early fetal hematopoiesis. This approach offers a valuable tool for studying complex cellular interactions in biological systems, providing new insights into hematopoietic niche organization during development.

Evaluating the ability of cytotoxic T lymphocytes (CTLs) to eliminate tumor cells is crucial, for instance, to predict the efficiency of cell therapy in personalized medicine. However, the destruction of a tumor by CTLs involves CTL migration in the extra-tumoral environment, accumulation on the tumor, antigen recognition, and cooperation in killing the cancer cells. Therefore, identifying the limiting steps in this complex process requires spatio-temporal measurements of different cellular events over long periods. Here, we use a cancer-on-a-chip platform to evaluate the impact of adenomatous polyposis coli (APC) mutation on CTL migration and cytotoxicity against 3D tumor spheroids. The APC mutated CTLs are found to have a reduced ability to destroy tumor spheroids compared with control cells, even though APC mutants migrate in the extra-tumoral space and accumulate on the spheroids as efficiently as control cells. Once in contact with the tumor however, mutated CTLs display reduced engagement with the cancer cells, as measured by a metric that distinguishes different modes of CTL migration. Realigning the CTL trajectories around localized killing cascades reveals that all CTLs transition to high engagement in the 2 h preceding the cascades, which confirms that the low engagement is the cause of reduced cytotoxicity. Beyond the study of APC mutations, this platform offers a robust way to compare cytotoxic cell efficiency of even closely related cell types, by relying on a multiscale cytometry approach to disentangle complex interactions and to identify the steps that limit the tumor destruction.

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: During embryogenesis, the hematopoietic system is formed by waves of progenitors that arise from the yolk sac (YS) and dorsal aorta (DA). These waves converge to the fetal liver (FL), where they display distinct dynamics of proliferation and differentiation. New evidence by ourselves and others show that YS, but not hematopoietic stem cell (HSC)-derived progenitors, drives hematopoiesis during the fetal period. Aims: Our aim is to identify how these two progenitor types respond to environmental cues to understand embryonic hematopoiesis. Methods: We evaluated the FL stromal compartment by spectral flow cytometry and single-cell gene expression, from mid-to-late gestation. We performed 3D immunofluorescence of the FL to identify the different stromal and hematopoietic populations and developed a cell-segmentation pipeline to extract single-cell positions and perform neighborhood analysis, resulting in a network representation of the FL. For the localization of cytokine expressing cells we used Kitl- and Cxcl12-reporter mice. Results: Here, we analyzed the FL microenvironment and found that at early stages both YS and DA-derived progenitors accumulate at the periphery of the FL where Kitl is mainly produced by mesothelial cells and hepatoblasts. Also, whereas certain hematopoietic cytokines (Csf1, Tpo and Kitl) are expressed by multiple cell types, Epo and Il7 are exclusively expressed by hepatoblasts, suggesting a critical role for these cells. Cytokine levels increase from E12 to E14 and decrease thereafter, indicating that the microenvironment is most supportive for hematopoiesis at earlier stages. At E14, while YS-derived progenitors remain concentrated at the periphery, DA-derived progenitors spread throughout the parenchyma in response to increased Cxcl12 levels in perivascular cells, compatible with their later migration to the bone marrow. Summary/Conclusion: Overall, we identified a new component of the FL microenvironment: the mesothelium and the periphery region. We hypothesize that low cytokine levels accommodate both the differentiation of YS-progenitors and the expansion of the HSC compartment due to their distinct sensitivity to environmental cues. Keywords: Fetal, Hematopoietic stem and progenitor cells, Hematopoietic microenvironment

During embryogenesis, yolk-sac and intra-embryonic-derived hematopoietic progenitors, comprising the precursors of adult hematopoietic stem cells, converge into the fetal liver. With a new staining strategy, we defined all non-hematopoietic components of the fetal liver and found that hepatoblasts are the major producers of hematopoietic growth factors. We identified mesothelial cells, a novel component of the stromal compartment, producing Kit ligand, a major hematopoietic cytokine. A high-definition imaging dataset analyzed using a deep-learning based pipeline allowed the unambiguous identification of hematopoietic and stromal populations, and enabled determining a neighboring network composition, at the single cell resolution. Throughout active hematopoiesis, progenitors preferentially associate with hepatoblasts, but not with stellate or endothelial cells. We found that, unlike yolk sac-derived progenitors, intra-embryonic progenitors respond to a chemokine gradient created by CXCL12-producing stellate cells. These results revealed that FL hematopoiesis is a spatiotemporal dynamic process, defined by an environment characterized by low cytokine concentrations.

The maintenance of neural stem cells (NSCs) in the adult brain depends on their activation frequency and division mode. Using long-term intravital imaging of NSCs in the zebrafish adult telencephalon, we reveal that apical surface area and expression of the Notch ligand DeltaA predict these NSC decisions. deltaA-negative NSCs constitute a bona fide self-renewing NSC pool and systematically engage in asymmetric divisions generating a self-renewing deltaAneg daughter, which regains the size and behavior of its mother, and a neurogenic deltaApos daughter, eventually engaged in neuronal production following further quiescence-division phases. Pharmacological and genetic manipulations of Notch, DeltaA, and apical size further show that the prediction of activation frequency by apical size and the asymmetric divisions of deltaAneg NSCs are functionally independent of Notch. These results provide dynamic qualitative and quantitative readouts of NSC lineage progression in vivo and support a hierarchical organization of NSCs in differently fated subpopulations.

Microscopy techniques and image segmentation algorithms have improved dramatically this decade, leading to an ever increasing amount of biological images and a greater reliance on imaging to investigate biological questions. This has created a need for methods to extract the relevant information on the behaviors of cells and their interactions, while reducing the amount of computing power required to organize this information.This task can be performed by using a network representation in which the cells and their properties are encoded in the nodes, while the neighborhood interactions are encoded by the links. Here, we introduce Griottes, an open-source tool to build the "network twin" of 2D and 3D tissues from segmented microscopy images. We show how the library can provide a wide range of biologically relevant metrics on individual cells and their neighborhoods, with the objective of providing multi-scale biological insights. The library's capacities are demonstrated on different image and data types.This library is provided as an open-source tool that can be integrated into common image analysis workflows to increase their capacities.