Expression of the pre-T cell receptor α (pTα) gene has been exploited in previous studies as a molecular marker to identify tiny cell populations in bone marrow (BM) and blood that were suggested to contain physiologically relevant thymus settling progenitors (TSPs). But to what extent these cells genuinely contribute to thymopoiesis has remained obscure. We have generated a novel pTα(iCre) knockin mouse line and performed lineage-tracing experiments to precisely quantitate the contribution of pTα-expressing progenitors to distinct differentiation pathways and to the genealogy of mature hematopoietic cells under physiological in vivo conditions. Using these mice in combination with fluorescent reporter strains, we observe highly consistent labeling patterns that identify pTα expression as a faithful molecular marker of T lineage commitment. Specifically, the fate of pTα-expressing progenitors was found to include all αβ and most γδ T cells but, in contrast to previous assumptions, to exclude B, NK, and thymic dendritic cells. Although we could detect small numbers of T cell progenitors with a history of pTα expression in BM and blood, our data clearly exclude these populations as physiologically important precursors of thymopoiesis and indicate that they instead belong to a pathway of T cell maturation previously defined as extrathymic.

T and innate lymphoid cells (ILCs) share some aspects of their developmental programs. However, although Notch signaling is strictly required for T cell development, it is dispensable for fetal ILC development. Constitutive activation of Notch signaling, at the common lymphoid progenitor stage, drives T cell development and abrogates ILC development by preventing Id2 expression. By combining single-cell transcriptomics and clonal culture strategies, we characterize two heterogeneous α4β7-expressing lymphoid progenitor compartments. αLP1 (Flt3(+)) still retains T cell potential and comprises the global ILC progenitor, while αLP2 (Flt3(-)) consists of ILC precursors that are primed toward the different ILC lineages. Only a subset of αLP2 precursors is sensitive to Notch signaling required for their proliferation. Our study identifies, in a refined manner, the diversity of transitional stages of ILC development, their transcriptional signatures, and their differential dependence on Notch signaling.

Hematopoietic stem cells (HSCs) self-renew in bone marrow niches formed by mesenchymal progenitors and endothelial cells expressing the chemokine CXCL12, but whether a separate niche instructs multipotent progenitor (MPP) differentiation remains unclear. We show that MPPs resided in HSC niches, where they encountered lineage-instructive differentiation signals. Conditional deletion of the chemokine receptor CXCR4 in MPPs reduced differentiation into common lymphoid progenitors (CLPs), which decreased lymphopoiesis. CXCR4 was required for CLP positioning near Interleukin-7+ (IL-7) cells and for optimal IL-7 receptor signaling. IL-7+ cells expressed CXCL12 and the cytokine SCF, were mesenchymal progenitors capable of differentiation into osteoblasts and adipocytes, and comprised a minor subset of sinusoidal endothelial cells. Conditional Il7 deletion in mesenchymal progenitors reduced B-lineage committed CLPs, while conditional Cxcl12 or Scf deletion from IL-7+ cells reduced HSC and MPP numbers. Thus, HSC maintenance and multilineage differentiation are distinct cell lineage decisions that are both controlled by HSC niches.

The Kit receptor functions in hematopoiesis, lymphocyte development, gastrointestinal tract motility, melanogenesis, and gametogenesis. To investigate the roles of different Kit signaling pathways in vivo, we have generated knock-in mice in which docking sites for PI 3-kinase (KitY719) or Src kinase (KitY567) have been mutated. Whereas steady-state hematopoiesis is normal in KitY719F/Y719F and KitY567F/Y567F mice, lymphopoiesis is affected differentially. The KitY567F mutation, but not the KitY719F mutation, blocks pro T cell and pro B cell development in an age-dependent manner. Thus, the Src family kinase, but not the PI 3-kinase docking site in Kit, mediates a critical signal for lymphocyte development. In agreement with these results, treatment of normal mice with the Kit tyrosine kinase inhibitor imatinib (Gleevec) leads to deficits in pro T and pro B cell development, similar to those seen in KitY567F/Y567F and KitW/W mice. The two mutations do not affect embryonic gametogenesis but the KitY719F mutation blocks spermatogenesis at the spermatogonial stages and in contrast the KitY567F mutation does not affect this process. Therefore, Kit-mediated PI 3-kinase signaling and Src kinase family signaling is highly specific for different cellular contexts in vivo.

Regulation of lineage biases in hematopoietic stem and progenitor cells (HSPCs) is pivotal for balanced hematopoietic output. However, little is known about the mechanism behind lineage choice in HSPCs. Here, we show that messenger RNA (mRNA) decay factors regnase-1 (Reg1; Zc3h12a) and regnase-3 (Reg3; Zc3h12c) are essential for determining lymphoid fate and restricting myeloid differentiation in HSPCs. Loss of Reg1 and Reg3 resulted in severe impairment of lymphopoiesis and a mild increase in myelopoiesis in the bone marrow. Single-cell RNA sequencing analysis revealed that Reg1 and Reg3 regulate lineage directions in HSPCs via the control of a set of myeloid-related genes. Reg1- and Reg3-mediated control of mRNA encoding Nfkbiz, a transcriptional and epigenetic regulator, was essential for balancing lymphoid/myeloid lineage output in HSPCs in vivo. Furthermore, single-cell assay for transposase-accessible chromatin sequencing analysis revealed that Reg1 and Reg3 control the epigenetic landscape on myeloid-related gene loci in early stage HSPCs via Nfkbiz. Consistently, an antisense oligonucleotide designed to inhibit Reg1- and Reg3-mediated Nfkbiz mRNA degradation primed hematopoietic stem cells toward myeloid lineages by enhancing Nfkbiz expression. Collectively, the collaboration between posttranscriptional control and chromatin remodeling by the Reg1/Reg3-Nfkbiz axis governs HSPC lineage biases, ultimately dictating the fate of lymphoid vs myeloid differentiation.