Definitive Hematopoiesis in the Yolk Sac Emerges from Wnt-Responsive Hemogenic Endothelium Independently of Circulation and Arterial Identity

Adult-repopulating hematopoietic stem cells (HSCs) emerge in low numbers in the midgestation mouse embryo from a subset of arterial endothelium, through an endothelial-to-hematopoietic transition. HSC-producing arterial hemogenic endothelium relies on the establishment of embryonic blood flow and arterial identity, and requires β-catenin signaling. Specified prior to and during the formation of these initial HSCs are thousands of yolk sac-derived erythro-myeloid progenitors (EMPs). EMPs ensure embryonic survival prior to the establishment of a permanent hematopoietic system, and provide subsets of long-lived tissue macrophages. While an endothelial origin for these HSC-independent definitive progenitors is also accepted, the spatial location and temporal output of yolk sac hemogenic endothelium over developmental time remain undefined. We performed a spatiotemporal analysis of EMP emergence, and document the morphological steps of the endothelial-to-hematopoietic transition. Emergence of rounded EMPs from polygonal clusters of Kit(+) cells initiates prior to the establishment of arborized arterial and venous vasculature in the yolk sac. Interestingly, Kit(+) polygonal clusters are detected in both arterial and venous vessels after remodeling. To determine whether there are similar mechanisms regulating the specification of EMPs with other angiogenic signals regulating adult-repopulating HSCs, we investigated the role of embryonic blood flow and Wnt/β-catenin signaling during EMP emergence. In embryos lacking a functional circulation, rounded Kit(+) EMPs still fully emerge from unremodeled yolk sac vasculature. In contrast, canonical Wnt signaling appears to be a common mechanism regulating hematopoietic emergence from hemogenic endothelium. These data illustrate the heterogeneity in hematopoietic output and spatiotemporal regulation of primary embryonic hemogenic endothelium

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Emergence and Regulation of Hemogenic Endothelium in the Mammalian Yolk Sac

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology and Laboratory Medicine, 2015.Hematopoietic stem cells are specified during embryogenesis from a specialized population of endothelium, termed hemogenic endothelium, which resides in major embryonic arteries and requires the hematopoietic transcription factor Runx1. Despite the established presence of these functional hematopoietic stem cells in the midgestation mouse embryo, embryonic survival is dependent on the earlier emergence and differentiation of a transient wave of yolk sac-derived erythro-myeloid progenitors. Previously, our laboratory confirmed that erythro-myeloid progenitors preferentially emerge from murine embryonic stem cell cultures. Conversely, transplantable hematopoietic stem cells have not yet been derived from embryonic stem cells, indicating the need to better understand the regulation of hematopoietic potential during development. Here, we define the cellular origin of erythro-myeloid progenitors, and begin to address mechanisms regulating their specification of during embryogenesis. We confirmed reports that erythro-myeloid progenitors require Runx1 for formation, and immunohistochemical studies strongly suggest they are derived from hemogenic endothelium. Subsequently, we examined hematopoietic emergence in Runx1+/- yolk sacs, which had reported alterations in hematopoietic potential, and found that reduced Runx1 dosage both delays the emergence of erythro-myeloid potential from hemogenic endothelium and results in increased proliferative potential. In the aorta, the specification of hematopoietic stem cell-producing hemogenic endothelium is dependent on embryonic circulation and β-catenin signaling. Interestingly, in the yolk sac, we found that hemogenic endothelium does not appear to be restricted to arterial vasculature, and analysis of circulation-deficient yolk sacs revealed normal cluster morphology and location, indicating the endothelial-tohematopoietic transition can occur in vivo without blood flow. Conversely, however, exogenous activation of β-catenin signaling in whole yolk sacs increased hematopoietic colony-forming activity. Subsequent in vitro assays and analysis of β-catenin reporter yolk sacs implicate a role for β-catenin signaling in hemogenic endothelium. Therefore, while emergence of erythro-myeloid progenitors from hemogenic endothelium is not dependent on blood flow or arterial identity, β-catenin appears to play a common role in hematopoietic specification from endothelium. Together, these data indicate the presence of heterogeneous populations of hemogenic endothelia in the mammalian conceptus, and highlight the need to understand developmental cues that influence the hematopoietic potential of each population in order to efficiently derive them in vitro

Distinct Sources of Hematopoietic Progenitors Emerge before HSCs and Provide Functional Blood Cells in the Mammalian Embryo

Hematopoietic potential arises in mammalian embryos before adult-repopulating hematopoietic stem cells (HSCs). At embryonic day 9.5 (E9.5), we show the first murine definitive erythro-myeloid progenitors (EMPs) have an immunophenotype distinct from primitive hematopoietic progenitors, maturing megakaryocytes and macrophages, and rare B cell potential. EMPs emerge in the yolk sac with erythroid and broad myeloid, but not lymphoid, potential. EMPs migrate to the fetal liver and rapidly differentiate, including production of circulating neutrophils by E11.5. Although the surface markers, transcription factors, and lineage potential associated with EMPs overlap with those found in adult definitive hematopoiesis, they are present in unique combinations or proportions that result in a specialized definitive embryonic progenitor. Furthermore, we find that embryonic stem cell (ESC)-derived hematopoiesis recapitulates early yolk sac hematopoiesis, including primitive, EMP, and rare B cell potential. EMPs do not have long-term potential when transplanted in immunocompromised adults, but they can provide transient adult-like RBC reconstitution

A systems biology pipeline identifies regulatory networks for stem cell engineering

A major challenge for stem cell engineering is achieving a holistic understanding of the molecular networks and biological processes governing cell differentiation. To address this challenge, we describe a computational approach that combines gene expression analysis, previous knowledge from proteomic pathway informatics and cell signaling models to delineate key transitional states of differentiating cells at high resolution. Our network models connect sparse gene signatures with corresponding, yet disparate, biological processes to uncover molecular mechanisms governing cell fate transitions. This approach builds on our earlier CellNet and recent trajectory-defining algorithms, as illustrated by our analysis of hematopoietic specification along the erythroid lineage, which reveals a role for the EGF receptor family member, ErbB4, as an important mediator of blood development. We experimentally validate this prediction and perturb the pathway to improve erythroid maturation from human pluripotent stem cells. These results exploit an integrative systems perspective to identify new regulatory processes and nodes useful in cell engineering.National Institute of General Medical Sciences (NIGMS) (Grant R01-GM081336)National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant R24-DK092760)National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R24-DK49216