Mds1 CreERT2-Based Lineage-Tracing Reveals Increasing Contributions of HSCs to Fetal Hematopoiesis and to Adult Tissue-Resident Macrophages in the Marrow

The ontogeny of the hematopoietic system consists of two broad programs. The first, an HSC-independent program, consists of overlapping waves of primitive, erythro-myeloid (EMP), and some lymphoid progenitors. HSC-independent hematopoiesis is required for normal fetal development, and provides self-renewing tissue-resident macrophage populations that persist in the adult. This is followed by the emergence of an HSC-dependent program that arises from arterial vessels within the body of the embryo. The overlapping emergence and lineage output of HSC-independent and HSC-derived hematopoiesis raises important questions regarding the identity and potential functional differences of their mature progeny. However, the transition from HSC-independent to HSC-derived hematopoiesis in the murine fetus remains incompletely characterized, particularly since the maturing erythroid, megakaryocytic and myeloid progeny of EMP and HSCs are currently not easily distinguishable. Additionally, lineage-tracing approaches have been challenging because they have relied largely on the temporal induction of promoters that are expressed both in HSC-independent progenitors and in HSCs, which have significant temporal overlap in their developmental emergence and result in incomplete or in mixed labeling. To help resolve this question, we have developed Mds1 CreERT2 mice, utilizing the first transcription start site of MECOM gene, which is expressed in HSC and emerging HSC (Yuasa et al., 2005 EMBO; Hou et al. 2020 Cell Research; Zhu et al. 2020, Blood). When mated with Rosa-YFP reporter mice and induced at E9.5 with tamoxifen, this construct lineage-traces pre-HSCs present in the E11.5 AGM region, as well as HSCs in the fetal liver and adult marrow. Importantly, no labeling of primitive erythroid cells, primitive macrophage-derived microglia, EMP, or EMP-derived cells in the E11.5 or E12.5 fetal liver was detected with tamoxifen induction at either E9.5 or E8.5. Analysis of E9.5 tamoxifen-treated Mds1 CreERT2Rosa26 LSL-YFP embryos indicates that HSCs have begun to generate small numbers of differentiating erythroid, myeloid and lymphoid progeny in the liver between E12.5 and E14.5. By E16.5, a significant proportion of differentiating erythroid, myeloid and B-cell lineage cells in the liver are HSC-derived, and HSCs have now begun to contribute erythroid and myeloid cells to the rapidly expanding pool of circulating blood cells. In the adult, we found increasing contributions of HSCs to macrophages in liver, lung and kidney. Interestingly, the majority of F4/80+ cells in the adult bone marrow and spleen were also lineage-traced in these mice. Thus, HSCs ultimately provide the majority of adult marrow macrophages that go on to self-maintain in the adult marrow (Hashimoto et al., 2013, Immunity). The Mds1 CreERT2 mouse model will serve as a useful to deconvolute the complexity of hematopoiesis as it unfolds in the embryo and functions postnatally. Disclosures Palis: Rubius Therapeutics: Consultancy.

Fetal Hematopoiesis is Driven by Privileged Expansion and Differentiation of Yolk Sac-Derived Erythro-Myeloid Progenitors

Most blood and immune cells are produced by Hematopoietic Stem Cells (HSC) throughout life. However, several tissue resident immune populations can only be generated from developmentally restricted progenitors. This questions to what extent fetal HSC differentiate in utero, implicating an essential role for HSC-independent progenitors in supporting embryonic viability and innate immunity in the perinatal period. Among them, Erythro-Myeloid Progenitors (EMP) emerge from the extraembryonic yolk sac prior to HSC and their progeny (resident macrophages and skin mast cells) persist in adulthood. Here, we showed that HSC contributed minimally to fetal myelopoiesis as we exposed a developmentally-restricted privilege for erythro-myeloid differentiation from EMP in the fetal liver. EMP-derived myeloid progenitors displayed distinct molecular features and were functionally inequivalent to fetal HSC-derived counterparts. These findings inform future studies of HSC-dependent and HSC-independent hematopoiesis in view of neonatal immunity and pediatric blood disorders for which the cell of origin is poorly understood.

Towards prevention of childhood ALL by early-life immune training

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common form of childhood cancer. Chemotherapy is associated with life-long health sequelae and fails in approximately 20% of cases. Thus, prevention of leukemia would be preferable to treatment. Childhood leukemia frequently starts before birth, during fetal hematopoiesis. A first genetic hit (e.g. the ETV6-RUNX1 gene fusion) leads to the expansion of pre-leukemic B-cell clones, which are detectable in healthy newborn cord blood (up to 5%). These pre-leukemic clones give rise to clinically overt leukemia in only about 0.2% of carriers. Experimental evidence suggests that a major driver of conversion from the pre-leukemic to the leukemic state is exposure to immune challenges. Novel insights have shed light on immune host responses and how they shape the complex interplay between (A) inherited or acquired genetic predispositions, (B) exposure to infection, and (C) abnormal cytokine release from immunologically untrained cells. Here, we integrate the recently emerging concept of "trained immunity" into existing models of childhood BCP-ALL and suggest future avenues towards leukemia prevention.

Maternal Obesity Dysregulates Fetal Hematopoietic Stem and Progenitor Cell Development in Rhesus Macaques

ABSTRACTInfants from obese moms have an increased susceptibility to immune dysregulation. However, the mechanisms by which maternal obesity alters fetal hematopoiesis remain largely unknown. Here, we determined the impact of maternal consumption of an obesogenic western-style diet (WSD) on hematopoietic development in fetal rhesus macaques using a combination of phenotypic, functional, and genomic assays. We demonstrate that maternal WSD resulted in accelerated fetal growth and altered fetal hematopoiesis. Specifically, single-cell RNA sequencing analysis of fetal bone marrow HSPCs showed that maternal WSD altered the transcriptional program of the common lymphoid progenitors and decreased the frequencies of bone marrow B-cells and NK-cells. Despite an expansion of monocyte progenitors in FBM, fetal blood monocytes from the WSD group demonstrated a blunted response to bacterial lipopolysaccharide. Furthermore, maternal WSD led to poor engraftment of fetal HSPCs in nonlethally irradiated immunodeficient NOD/SCID/IL2rγ-/-mice. Collectively, this study demonstrates that maternal WSD dysregulates fetal HSPC development.

Fetal-Derived Immune Cells at the Roots of Lifelong Pathophysiology

Tissue-resident innate immune cells exert a wide range of functions in both adult homeostasis and pathology. Our understanding of when and how these cellular networks are established has dramatically changed with the recognition that many lineages originate at least in part from fetal sources and self-maintain independently from hematopoietic stem cells. Indeed, fetal-derived immune cells are found in most organs and serous cavities of our body, where they reside throughout the entire lifespan. At the same time, there is a growing appreciation that pathologies manifesting in adulthood may be caused by adverse early life events, a concept known as “developmental origins of health and disease” (DOHaD). Yet, whether fetal-derived immune cells are mechanistically involved in DOHaD remains elusive. In this review, we summarize our knowledge of fetal hematopoiesis and its contribution to adult immune compartments, which results in a “layered immune system.” Based on their ontogeny, we argue that fetal-derived immune cells are prime transmitters of long-term consequences of prenatal adversities. In addition to increasing disease susceptibility, these may also directly cause inflammatory, degenerative, and metabolic disorders. We explore this notion for cells generated from erythro-myeloid progenitors (EMP) produced in the extra-embryonic yolk sac. Focusing on macrophages and mast cells, we present emerging evidence implicating them in lifelong disease by either somatic mutations or developmental programming events resulting from maternal and early environmental perturbations.

YY1 Promotes SCF/c-Kit Signaling and HSC Self-Renewal in Fetal Hematopoiesis

Fetal hematopoietic stem cells (HSCs) exhibit markedly different properties as compared to adult HSCs including cell surface marker expression, proliferation state and repopulation capacity. Changes of HSC activity in postnatal mice is defined by a process of decreasing in cell cycle and entering into quiescence. Yin Yang 1 (YY1) is a ubiquitous transcription factor and mammalian Polycomb Group (PcG) Protein with important functions to regulate embryonic development, lineage differentiation and cell proliferation. While homozygote deletion of YY1 in mice results in lethality at the peri-implantation stage, heterozygote deletion of YY1 causes severe developmental defects. By conditionally deleting YY1 in adult hematopoietic system, our previous study showed that YY1 is an essential regulator for adult hematopoiesis by promoting HSC long-term self-renewal and maintaining adult HSC quiescence. In contrast to adult HSCs, in which quiescence is a fundamental characteristic, over 95% of fetal HSCs are in an active cell cycle to rapidly generate homeostatic levels of blood cells for oxygen transport and immune system development in the growing organism. Herein, we assessed whether YY1 was also required for maintaining fetal HSC pool and regulating fetal HSC functions, and what was the underlying mechanism by which YY1 regulated fetal HSCs. To test how loss-of-function of YY1 impacted fetal hematopoiesis, Yy1f/f mice in which the Yy1 promoter region and exon 1 are flanked by loxP sites, were crossed to Vav-Cre mice to generate heterozygous Yy1f/+ Vav-cre mice. The Vav promoter drives Cre recombinase expression specifically in fetal liver hematopoietic cell starting at day E11.5. Yy1f/+ Vav-cre mice were then subsequently bred with Yy1f/f mice to generate homozygous Yy1f/f Vav-cre mice. Among 141 pups resulting from breeding Yy1f/fto Yy1f/+ Vav-Cre, only 7 were Yy1f/f Vav-Cre (n=7) and was significantly lower than the estimated number (n=35) according to the Mendelian ratio (P<0.05). All Yy1f/f Vav-Cre pups died within 72 hours after birth, which supported essential role of YY1 in fetal hematopoiesis and survival. At E14.5 of fetal development, Yy1f/f Vav-Cre fetuses had reduced numbers of hematopoietic stem and progenitor cells in the liver. In addition, YY1 deficient fetal HSCs failed to self-renew in primary and secondary bone marrow transplantation assays. Colony formation assay showed that fetal liver cells from Yy1f/f Vav-Cre mice failed to form CFU-GEMM, CFU-GU and BFU-E compared to Vav-Cre control. While YY1 promotes SCF/c-Kit signaling in adult HSCs, it does not impact c-Kit cell surface expression in early T cell progenitors (unpublished data). To assess YY1 impact on SCF/c-Kit axle in fetal HSCs, c-Kit transcript level, c-Kit median fluorescence intensity and phosphorylated AKT were measured. Similar as its function in adult HSCs, YY1 deficient fetal HSCs had decreased Kit transcript expression, decreased c-Kit cell surface expression and decreased SCF/c-Kit signaling. Our results supported that YY1 is required for maintaining a continuous pool of HSCs in fetal liver and is critical for fetal HSC long-term self-renewal and differentiation. Similar as its function in adult HSCs, YY1 promotes SCF/c-Kit signaling in fetal HSCs. Disclosures No relevant conflicts of interest to declare.

The Role of DOT1L Methyltransferase Activity in Fetal Hematopoiesis

ABSTRACTEarly mammalian erythropoiesis requires the DOT1L methyltransferase. We demonstrated that loss of DOT1L in mutant mice resulted in lethal anemia during midgestation. The molecular mechanisms by which DOT1L regulates embryonic erythropoiesis have not yet been elucidated. In this study, a methyltransferase mutant mouse line (Dot1L-MM) was generated to determine whether the methyltransferase activity of DOT1L is essential for erythropoiesis. Dot1L-MM mice displayed embryonic lethality between embryonic days 10.5 and 13.5, similar to Dot1lL knockout (Dot1L-KO) mice. However, when examined at E10.5, unlike the Dot1L-KO, Dot1L-MM embryos did not exhibit evidence of anemia. In ex vivo hematopoietic differentiation cultures, Dot1L-KO and Dot1L-MM yolk sac (YS) cells both formed reduced numbers of myeloid, and mixed hematopoietic colonies. Erythroid colonies were able to be formed in numbers equal to wildtype embryos. Extensively self-renewing erythroblast (ESRE) cultures were established using YS cells from E10.5 embryos. Dot1L-KO and Dot1L-MM cells expanded significantly less than wild-type cells and exhibited increased cell death. Strikingly, Dot1L-KO and Dot1L-MM cells of YS origin exhibited profound genomic instability, implicating DOT1L methyltransferase activity in maintenance of the genome as well as viability of hematopoietic progenitors. Our results indicate that the methyltransferase activity of DOT1L plays an important role early murine hematopoiesis.

Sustained fetal hematopoiesis causes juvenile death from leukemia: evidence from a dual-age–specific mouse model

It is not clear whether disrupted age-specific hematopoiesis contributes to the complex manifestations in leukemia patients who carry identical mutations, particularly in pediatric and adult patients with similar clinical characteristics. By studying a dual-age–specific mouse model, we demonstrate that (1) loss of Pten during the fetal-to-adult hematopoiesis switch (hematopoiesis switch) causes sustained fetal hematopoiesis, resulting in death in juvenile leukemia; (2) myeloid-biased hematopoiesis in juvenile mice is associated with the sustained fetal properties of hematopoietic stem cells (HSCs); (3) the age specificity of juvenile myelomonocytic leukemia depends on the copy number of Pten and Nf1; (4) single-allelic Pten deletion during the hematopoiesis switch causes constitutive activation of MAPK in juvenile mice with Nf1 loss of heterozygosity (LOH); and (5) Nf1 LOH causes monocytosis in juvenile mice with Pten haploinsufficiency but does not cause lethality until adulthood. Our data suggest that 1 copy of Pten is sufficient to maintain an intact negative-feedback loop of the Akt pathway and HSC function in reconstitution, despite MAPK being constitutively activated in juvenile Pten+/ΔNf1LOH mice. However, 2 copies of Pten are required to maintain the integrity of the MAPK pathway in juvenile mice with Nf1 haploinsufficiency. Our data indicate that previous investigations of Pten function in wild-type mice may not reflect the impact of Pten loss in mice with Nf1 mutations or other genetic defects. We provide a proof of concept that disassociated age-specific hematopoiesis contributes to leukemogenesis and pediatric demise.