In vivo clonal tracking reveals evidence of haemangioblast and haematomesoblast contribution to yolk sac haematopoiesis

Abstract

During embryogenesis, haematopoietic and endothelial lineages emerge closely in time and space. It is thought that the first blood and endothelium derive from a common clonal ancestor, the haemangioblast. However, investigation of candidate haemangioblasts in vitro revealed the capacity for mesenchymal differentiation, a feature more compatible with an earlier mesodermal precursor. To date, no evidence for an in vivo haemangioblast has been discovered. Using single cell RNA-Sequencing and in vivo cellular barcoding, we have unravelled the ancestral relationships that give rise to the haematopoietic lineages of the yolk sac, the endothelium, and the mesenchyme. We show that the mesodermal derivatives of the yolk sac are produced by three distinct precursors with dual-lineage outcomes: the haemangioblast, the mesenchymoangioblast, and a previously undescribed cell type: the haematomesoblast. Between E5.5 and E7.5, this trio of precursors seeds haematopoietic, endothelial, and mesenchymal trajectories.

Developmental regulation of primitive erythropoiesis

“…The advent of single-cell transcriptomics with increasing read depths together with in-vivo barcoding methods has allowed investigators to revisit the question of the origin of primitive erythroid lineage in the unperturbed murine embryo. These studies confirm the results of prior ex-vivo colony-forming assays regarding a hemangioblast source and extends them with the identification of a mesodermal progenitor with mesenchymal potential [11 ▪▪ ]. The independent existence of these mesodermal progenitors and the molecular machinery that governs their erythroid commitment await further investigation, ideally combined with precise conditional knockout of key factors driven by new cell-type-specific transcription factors that emerge from the single-cell studies.…”

“…Another recent study analyzing single-cell RNA sequencing of E7.25 and E7.75 mouse embryos identified a sizeable population of hematoendothelial precursors with an unexpected mesenchymal signature that was abruptly downregulated by E8.5 [11 ▪▪ ]. Tracing analysis of unperturbed hematopoiesis by a cleverly designed inducible Cre-LoxP-based cellular barcoding system [12] in a newly generated mouse line revealed, apart from the existence of hemangioblasts, the existence of another pathway contributing to blood and mesenchymal elements, termed hematomesoblast (Fig.…”

“…Decades of research has resulted in ever improving differentiation protocols, which now employ signaling factors identified in gastrulating embryos to drive primitive versus definitive erythroid fates [25]. For example, treatment of human ESCs with Activin A, BMP4, and FGF2 induces KDR + CD235a + mesoderm (of note, CD235a is only expressed in human, not mouse, mesoderm [11 ▪▪ ]) in embryoid bodies that upon dissociation and re-plating in media containing FGF2, VEGF, and multiple cytokines results in CD43 + primitive erythroid progenitors that form colonies of embryonic globin ( HBE )-expressing cells [26]. Previous work had shown that chemical inhibition of the Wnt-signaling pathway can greatly increase the generation of CD43 + EryP-CFC [25].…”

APax3lineage gives rise to transient haematopoietic progenitors

“…As Pax3 is an established marker of paraxial mesoderm and neural crest, both populations might be considered as potential sources of Pax3 lineage haematopoietic progenitors. In particular, it is conceivable that these progenitors might arise from a subset of paraxial mesoderm-derived endothelial cells with haemogenic potential, in a process analogous to that described for extra-embryonic mesoderm-derived haemogenic endothelium in the yolk sac or lateral plate mesoderm-derived haemogenic endothelium in the dorsal aorta 11,14,15 . Alternatively, Pax3 may be expressed in a distinct and hitherto unrecognised source of progenitors with haematopoietic potential that may or may not pass through an endothelial intermediate, in which case Pax3-mediated lineage tracing would be insufficient to attribute cellular origins to paraxial mesoderm.…”

“…During embryonic development, haematopoiesis occurs in temporally and spatially overlapping waves that originate from well-defined tissues 10 . Extra-embryonic mesoderm gives rise to yolk sac haemogenic endothelium that transitions into pro-definitive haematopoietic progenitors, including erythro-myeloid progenitors (EMPs) and lymphomyeloid progenitors (LMPs) [10][11][12] . By contrast, intra-embryonic lateral plate mesoderm produces haemogenic endothelium in the dorsal aorta that transitions into definitive haematopoietic stem cells (HSCs) 10,13,14 .…”