Generation of specialized blood vessels via lymphatic transdifferentiation.

The lineage and developmental trajectory of a cell are key determinants of cellular identity. In the vascular system, endothelial cells (ECs) of blood and lymphatic vessels differentiate and specialize to cater to the unique physiological demands of each organ1,2. Although lymphatic vessels were shown to derive from multiple cellular origins, lymphatic ECs (LECs) are not known to generate other cell types3,4. Here we use recurrent imaging and lineage-tracing of ECs in zebrafish anal fins, from early development to adulthood, to uncover a mechanism of specialized blood vessel formation through the transdifferentiation of LECs. Moreover, we demonstrate that deriving anal-fin vessels from lymphatic versus blood ECs results in functional differences in the adult organism, uncovering a link between cell ontogeny and functionality. We further use single-cell RNA-sequencing analysis to characterize the different cellular populations and transition states involved in the transdifferentiation process. Finally, we show that, similar to normal development, the vasculature is rederived from lymphatics during anal-fin regeneration, demonstrating that LECs in adult fish retain both potency and plasticity for generating blood ECs. Overall, our research highlights an innate mechanism of blood vessel formation through LEC transdifferentiation, and provides in vivo evidence for a link between cell ontogeny and functionality in ECs.

A non-classical monocyte-derived macrophage subset provides a splenic replication niche for intracellular Salmonella.

Interactions between intracellular bacteria and mononuclear phagocytes give rise to diverse cellular phenotypes that may determine the outcome of infection. Recent advances in single-cell RNA sequencing (scRNA-seq) have identified multiple subsets within the mononuclear population, but implications to their function during infection are limited. Here, we surveyed the mononuclear niche of intracellular Salmonella Typhimurium (S.Tm) during early systemic infection in mice. We described eclipse-like growth kinetics in the spleen, with a first phase of bacterial control mediated by tissue-resident red-pulp macrophages. A second phase involved extensive bacterial replication within a macrophage population characterized by CD9 expression. We demonstrated that CD9+ macrophages induced pathways for detoxificating oxidized lipids, that may be utilized by intracellular S.Tm. We established that CD9+ macrophages originated from non-classical monocytes (NCM), and NCM-depleted mice were more resistant to S.Tm infection. Our study defines macrophage subset-specific host-pathogen interactions that determine early infection dynamics and infection outcome of the entire organism.

VEGFC/FLT4-induced cell-cycle arrest mediates sprouting and differentiation of venous and lymphatic endothelial cells.

The formation of new vessels requires a tight synchronization between proliferation, differentiation, and sprouting. However, how these processes are differentially activated, often by neighboring endothelial cells (ECs), remains unclear. Here, we identify cell cycle progression as a regulator of EC sprouting and differentiation. Using transgenic zebrafish illuminating cell cycle stages, we show that venous and lymphatic precursors sprout from the cardinal vein exclusively in G1 and reveal that cell-cycle arrest is induced in these ECs by overexpression of p53 and the cyclin-dependent kinase (CDK) inhibitors p27 and p21. We further demonstrate that, in vivo, forcing G1 cell-cycle arrest results in enhanced vascular sprouting. Mechanistically, we identify the mitogenic VEGFC/VEGFR3/ERK axis as a direct inducer of cell-cycle arrest in ECs and characterize the cascade of events that render "sprouting-competent" ECs. Overall, our results uncover a mechanism whereby mitogen-controlled cell-cycle arrest boosts sprouting, raising important questions about the use of cell cycle inhibitors in pathological angiogenesis and lymphangiogenesis.

Synapsins regulate α-synuclein functions.

The normal function of α-synuclein (α-syn) remains elusive. Although recent studies suggest α-syn as a physiologic attenuator of synaptic vesicle (SV) recycling, mechanisms are unclear. Here, we show that synapsin-a cytosolic protein with known roles in SV mobilization and clustering-is required for presynaptic functions of α-syn. Our data offer a critical missing link and advocate a model where α-syn and synapsin cooperate to cluster SVs and attenuate recycling.

Using FRAP or FRAPA to Visualize the Movement of Fluorescently Labeled Proteins or Cellular Organelles in Live Cultured Neurons Transformed with Adeno-Associated Viruses.

Fluorescence recovery after photobleaching (FRAP) and fluorescence redistribution after photoactivation (FRAPA) are complementary methods used to gauge the movement of proteins or sub-resolution organelles within cells. Using these methods we can determine the nature of the movement of labeled particles, whether it is random, constrained, or active, the coefficient of diffusion if applicable, binding and unbinding constants, and the direction of active transport. These two techniques have been extensively utilized to probe the cell biology of neurons. A practical outline of FRAP and FRAPA in cultured neurons is presented, including the preparation of the neurons and their infection with adeno-associated viral vectors. Considerations in planning such experiments are provided.