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Current dogma asserts that the foetal liver (FL) is an expansion niche for recently specified haematopoietic stem cells (HSCs) during ontogeny. Indeed, between embryonic day of development (E)12.5 and E14.5, the number of transplantable HSCs in the murine FL expands from 50 to about 1,000. Here we used a non-invasive, multi-colour lineage tracing strategy to interrogate the embryonic expansion of murine haematopoietic progenitors destined to contribute to the adult HSC pool. Our data show that this pool of fated progenitors expands only two-fold during FL ontogeny. Although Histone2B-GFP retention in vivo experiments confirmed substantial proliferation of phenotypic FL-HSC between E12.5 and E14.5, paired-daughter cell assays revealed that many mid-gestation phenotypic FL-HSCs are biased to differentiate, rather than self-renew, relative to phenotypic neonatal and adult bone marrow HSCs. In total, these data support a model in which the FL-HSC pool fated to contribute to adult blood expands only modestly during ontogeny.

The terminal maturation of human erythroblasts requires significant changes in gene expression in the context of dramatic nuclear condensation. Defects in this process are associated with inherited anemias and myelodysplastic syndromes. The progressively dense appearance of the condensing nucleus in maturing erythroblasts led to the assumption that heterochromatin accumulation underlies this process, but despite extensive study, the precise mechanisms underlying this essential biologic process remain elusive. To delineate the epigenetic changes associated with the terminal maturation of human erythroblasts, we performed mass spectrometry of histone posttranslational modifications combined with chromatin immunoprecipitation coupled with high-throughput sequencing, Assay for Transposase Accessible Chromatin, and RNA sequencing. Our studies revealed that the terminal maturation of human erythroblasts is associated with a dramatic decline in histone marks associated with active transcription elongation, without accumulation of heterochromatin. Chromatin structure and gene expression were instead correlated with dynamic changes in occupancy of elongation competent RNA polymerase II, suggesting that terminal erythroid maturation is controlled largely at the level of transcription. We further demonstrate that RNA polymerase II “pausing” is highly correlated with transcriptional repression, with elongation competent RNA polymerase II becoming a scare resource in late-stage erythroblasts, allocated to erythroid-specific genes. Functional studies confirmed an essential role for maturation stage-specific regulation of RNA polymerase II activity during erythroid maturation and demonstrate a critical role for HEXIM1 in the regulation of gene expression and RNA polymerase II activity in maturing erythroblasts. Taken together, our findings reveal important insights into the mechanisms that regulate terminal erythroid maturation and provide a novel paradigm for understanding normal and perturbed erythropoiesis.

RationaleWhile animal model studies have extensively defined mechanisms controlling cell diversity in the developing mammalian lung, the limited data available from late stage human lung development represents a significant knowledge gap. The NHLBI Molecular Atlas of Lung Development Program (LungMAP) seeks to fill this gap by creating a structural, cellular and molecular atlas of the human and mouse lung.MethodsSingle cell RNA sequencing generated transcriptional profiles of 5500 cells obtained from two one-day old human lungs (born at gestational ages of 39 and 31 weeks) from the LungMAP Human Tissue Core Biorepository at the University of Rochester. Frozen single cell isolates were captured, and library preparation was completed on the Chromium 10X system. Data was analyzed in Seurat, and cellular annotation was performed using the ToppGene functional analysis tool. Single cell sequence data from 32000 postnatal day 1, 3, 7 and 10 mouse lung (n = 2 at each time point) cells generated by the LungMAP Research Center at Cincinnati Children’s Hospital and Medical Center, using Dropseq platform, was integrated with the human data. In situ hybridization was used to confirm the spatial location of cellular phenotypes.ResultsTranscriptional interrogation of donor newborn human lung cells identified distinct clusters representing multiple populations of epithelial, endothelial, fibroblasts, pericytes, smooth muscle, and immune cells and signature genes for each of these populations were identified. Computational integration of newborn human and postnatal mouse lung development cellular transcriptomes facilitated the identification of distinct cellular lineages among all the major cell types. Integration of the human and mouse cellular transcriptomes also demonstrated cell type-specific differences in developmental states of the newborn human lung cells. In particular, matrix fibroblasts could be separated into those representative of younger cells (n=393), or older cells (n=158).This is the first comprehensive molecular map of the cellular landscape of neonatal human lung, including biomarkers for cells at distinct states of development. Our results indicate that integrated single cell RNA profiling of human and mouse lung will help identify common and species-specific mechanisms of lung development and respiratory disease.

Myelodysplastic syndromes are clonal stem cell disorders associated with low blood counts and a high risk of leukemic transformation. Survival ranges from months to years and bone marrow transplantation remains the only curative option. Large sequencing studies have revealed that somatic mutations in epigenetic regulators are one of the most commonly affected pathways in MDS. DNMT3A, a de novo methyltransferase is mutated in ~10-15% of MDS patients. Murine models of DNMT3A loss have shown that hematopoietic stem cells favor self-renewal over differentiation. Loss of function mutations in DNMT3A can lead to reduction in methylation resulting in differentially methylated regions (DMRs). The effects of altered DNA methylation at promoters and enhancers on gene expression in DNMT3A mutant cells are well characterized but do not completely explain the gene expression patterns in these cells. Previous studies have shown that DNA methylation within intragenic regions affects mRNA splicing through interactions with RNA polymerase II and DNA binding proteins. Therefore, our study aimed to determine whether altered methylation caused by DNMT3A loss affects mRNA splicing and gene expression. To address this, we used CRISPR/Cas9 to genetically knockout DNMT3A in K562 cells. Findings were further evaluated using lineage- c-kit+ hematopoietic stem and progenitor cells (HSPC) from a DMNT3A conditional knock in mouse model of the most common MDS-associated DNMT3A point mutation, R878H. Our central hypothesis is that aberrant methylation caused by loss of function mutations in DNMT3A leads to changes in RNA splicing resulting in differential isoform expression of genes involved in stem cell self-renewal and hematopoiesis. Using a combination of RNA sequencing and whole genome bisulfite sequencing we found increased alternative splicing in DNMT3A mutant cells compared to wildtype cells. Integration of methylation and splicing revealed that 15-20 percent of the alternative splicing events were associated with changes in methylation. We found that many of the genes aberrantly spliced were involved in cell cycle regulation, transcriptional regulation, and hematopoietic stem cell differentiation. In addition, isoform usage analysis revealed that genes involved in RNA processing were perturbed. We identified similar splicing patterns and affected pathways in murine HSPCs and K562 human cells. Notably, the type of splicing events changed with age. Our results show that DNMT3A mutant cells have distinct alternative splicing events and suggest an aberrant epigenome can cause dysregulated gene expression through splicing. These findings shed light onto additional mechanisms by which epigenetic regulator mutations lead to MDS development. Citation Format: LaShanale Wallace, Ana Leal-Cervantes, Jacquelyn Myers, Yiping Fan, Koral Campbell, Juan Martin Barajas, Aparna Calindi, Chandra Rolle, Baranda Hansen, Shondra Miller, Esther Obeng. Alternative splicing in hematopoietic stem cells is affected by the loss of DNMT3A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2093.

Hematopoietic ontogeny unfolds in two broad programs- a complex hematopoietic stem cell (HSC)-independent program followed by an HSC-dependent program. We have previously determined that HSC-independent, multi-lineage “erythro-myeloid” progenitors (EMP) emerge from hemogenic endothelium in the yolk sac of the murine embryo and engraft the fetal liver to generate the first definitive erythroid, granulocyte, monocyte, and NK cells prior to the establishment of HSC-derived hematopoiesis. Analysis of single cell transcriptomic data from E9.5 EMP and from hematopoietic cells in the E12.5 fetal liver provides a unique opportunity to tie predictions of progenitor identity to subsequent differentiation states. We analyzed 2,959 kit+CD41+CD16/32+ yolk sac EMP and 7,495 fetal liver hematopoietic cells pooled from 30 E9.5 and 3 E12.5 embryos, respectively, using the 10X Genomics and inDrops platforms. Dimensionality reduction coupled with graph-based clustering and analysis of differentially expressed marker genes allowed us to presumptively identify erythroid-, megakaryocyte-, macrophage-, and myeloid-biased clusters both in EMP and in the E12.5 fetal liver. Functional colony-forming assays confirm the existence of primary myeloid- and erythroid-biased EMP in E9.5 embryos. E9.5 EMP also contain a cluster of innate lymphoid-biased progenitors, consistent with the recently identified NK cell lineage potential of EMP. Integration of EMP and fetal liver datasets further supported the concept that EMP consist of a surprising, early complexity of lineage-biased progenitors, whose differentiation continue in the fetal liver. Interestingly, EMP also contained a cluster of hemogenic endothelial-biased progenitors that were not found in the fetal liver, and which serve as a source of diverging erythroid/megakaryocyte and innate lymphoid/myeloid pseudotime trajectories. Taken together, our data support the concept that EMP emerge from hemogenic endothelium and begin to differentiate into multiple lineage-biased progenitors before engrafting the fetal liver to complete their differentiation along erythroid/megakaryocytic and innate immune cell paths to meet the needs of the developing embryo prior to the establishment of HSC-derived blood cell production.