Mapping human haematopoietic stem cells from haemogenic endothelium to birth.

The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites1. Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the aorta-gonad-mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of the HSC maturity markers CD133 (encoded by PROM1) and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ haemogenic endothelial cells, which arose from an IL33+ALDH1A1+ arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta-gonad-mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.

MLLT3 governs human haematopoietic stem-cell self-renewal and engraftment.

Limited knowledge of the mechanisms that govern the self-renewal of human haematopoietic stem cells (HSCs), and why this fails in culture, have impeded the expansion of HSCs for transplantation1. Here we identify MLLT3 (also known as AF9) as a crucial regulator of HSCs that is highly enriched in human fetal, neonatal and adult HSCs, but downregulated in culture. Depletion of MLLT3 prevented the maintenance of transplantable human haematopoietic stem or progenitor cells (HSPCs) in culture, whereas stabilizing MLLT3 expression in culture enabled more than 12-fold expansion of transplantable HSCs that provided balanced multilineage reconstitution in primary and secondary mouse recipients. Similar to endogenous MLLT3, overexpressed MLLT3 localized to active promoters in HSPCs, sustained levels of H3K79me2 and protected the HSC transcriptional program in culture. MLLT3 thus acts as HSC maintenance factor that links histone reader and modifying activities to modulate HSC gene expression, and may provide a promising approach to expand HSCs for transplantation.

Intestinal serotonin and fluoxetine exposure modulate bacterial colonization in the gut.

The gut microbiota regulates levels of serotonin (5-hydroxytryptamine (5-HT)) in the intestinal epithelium and lumen1-5. However, whether 5-HT plays a functional role in bacteria from the gut microbiota remains unknown. We demonstrate that elevating levels of intestinal lumenal 5-HT by oral supplementation or genetic deficiency in the host 5-HT transporter (SERT) increases the relative abundance of spore-forming members of the gut microbiota, which were previously reported to promote host 5-HT biosynthesis. Within this microbial community, we identify Turicibacter sanguinis as a gut bacterium that expresses a neurotransmitter sodium symporter-related protein with sequence and structural homology to mammalian SERT. T. sanguinis imports 5-HT through a mechanism that is inhibited by the selective 5-HT reuptake inhibitor fluoxetine. 5-HT reduces the expression of sporulation factors and membrane transporters in T. sanguinis, which is reversed by fluoxetine exposure. Treating T. sanguinis with 5-HT or fluoxetine modulates its competitive colonization in the gastrointestinal tract of antibiotic-treated mice. In addition, fluoxetine reduces the membership of T. sanguinis in the gut microbiota of conventionally colonized mice. Host association with T. sanguinis alters intestinal expression of multiple gene pathways, including those important for lipid and steroid metabolism, with corresponding reductions in host systemic triglyceride levels and inguinal adipocyte size. Together, these findings support the notion that select bacteria indigenous to the gut microbiota signal bidirectionally with the host serotonergic system to promote their fitness in the intestine.

Viral Capsid Trafficking along Treadmilling Tubulin Filaments in Bacteria.

Cargo trafficking along microtubules is exploited by eukaryotic viruses, but no such examples have been reported in bacteria. Several large Pseudomonas phages assemble a dynamic, tubulin-based (PhuZ) spindle that centers replicating phage DNA sequestered within a nucleus-like structure. Here, we show that capsids assemble on the membrane and then move rapidly along PhuZ filaments toward the phage nucleus for DNA packaging. The spindle rotates the phage nucleus, distributing capsids around its surface. PhuZ filaments treadmill toward the nucleus at a constant rate similar to the rate of capsid movement and the linear velocity of nucleus rotation. Capsids become trapped along mutant static PhuZ filaments that are defective in GTP hydrolysis. Our results suggest a transport and distribution mechanism in which capsids attached to the sides of filaments are trafficked to the nucleus by PhuZ polymerization at the poles, demonstrating that the phage cytoskeleton evolved cargo-trafficking capabilities in bacteria.

The Phage Nucleus and Tubulin Spindle Are Conserved among Large Pseudomonas Phages.

We recently demonstrated that the large Pseudomonas chlororaphis bacteriophage 201φ2-1 assembles a nucleus-like structure that encloses phage DNA and segregates proteins according to function, with DNA processing proteins inside and metabolic enzymes and ribosomes outside the nucleus. Here, we investigate the replication pathway of the Pseudomonas aeruginosa bacteriophages φKZ and φPA3. Bacteriophages φKZ and φPA3 encode a proteinaceous shell that assembles a nucleus-like structure that compartmentalizes proteins and DNA during viral infection. We show that the tubulin-like protein PhuZ encoded by each phage assembles a bipolar spindle that displays dynamic instability and positions the nucleus at midcell. Our results suggest that the phage spindle and nucleus play the same functional role in all three phages, 201φ2-1, φKZ, and φPA3, demonstrating that these key structures are conserved among large Pseudomonas phages.

Assembly of a nucleus-like structure during viral replication in bacteria.

We observed the assembly of a nucleus-like structure in bacteria during viral infection. Using fluorescence microscopy and cryo-electron tomography, we showed that Pseudomonas chlororaphis phage 201φ2-1 assembled a compartment that separated viral DNA from the cytoplasm. The phage compartment was centered by a bipolar tubulin-based spindle, and it segregated phage and bacterial proteins according to function. Proteins involved in DNA replication and transcription localized inside the compartment, whereas proteins involved in translation and nucleotide synthesis localized outside. Later during infection, viral capsids assembled on the cytoplasmic membrane and moved to the surface of the compartment for DNA packaging. Ultimately, viral particles were released from the compartment and the cell lysed. These results demonstrate that phages have evolved a specialized structure to compartmentalize viral replication.