A novel prospective isolation of murine fetal liver progenitors to study in utero hematopoietic defects.

Julia E Draper,Muhammad Z H Fadlullah,Wolfgang Breitwieser,Gillian Newton,Rahima Patel,Georges Lacaud,Valerie Kouskoff,Patrycja Sroczynska

doi:10.1371/journal.pgen.1007127

Julia E Draper, Muhammad Z H Fadlullah + Show 6 more

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https://doi.org/10.1371/journal.pgen.1007127

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Abstract

In recent years, highly detailed characterization of adult bone marrow (BM) myeloid progenitors has been achieved and, as a result, the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined. Fetal liver (FL) hematopoietic progenitor cells (HPCs) are poorly characterized in comparison, potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis. Numerous disorders, for example infant acute leukemias, have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets. We previously demonstrated that a Runx1 distal promoter (P1)-GFP::proximal promoter (P2)-hCD4 dual-reporter mouse (Mus musculus) model can be used to identify adult BM progenitor subsets with distinct lineage preferences. In this study, we undertook the characterization of the expression of Runx1-P1-GFP and P2-hCD4 in FL. Expression of P2-hCD4 in the FL immunophenotypic Megakaryocyte-Erythroid Progenitor (MEP) and Common Myeloid Progenitor (CMP) compartments corresponded to increased granulocytic/monocytic/megakaryocytic and decreased erythroid specification. Moreover, Runx1-P2-hCD4 expression correlated with several endogenous cell surface markers’ expression, including CD31 and CD45, providing a new strategy for prospective identification of highly purified fetal myeloid progenitors in transgenic mouse models. We utilized this methodology to compare the impact of the deletion of either total RUNX1 or RUNX1C alone and to determine the fetal HPCs lineages most substantially affected. This new prospective identification of FL progenitors therefore raises the prospect of identifying the underlying gene networks responsible with greater precision than previously possible.

Highlights

Definitive hematopoiesis is a complex, multistep process involving increasingly restrictive cell fate decisions by self-renewing, multipotent hematopoietic stem cells (HSCs)
The production of red blood cells, platelet-producing megakaryocytes, and immune response-directing granulocytes and monocytes is initiated at an early stage in the developing embryo and continues throughout life
We know that in the mouse embryo, specialized blood progenitor cells emerge in the fetal liver and produce mature blood cells in response to different cues

Summary

Introduction

Definitive hematopoiesis is a complex, multistep process involving increasingly restrictive cell fate decisions by self-renewing, multipotent hematopoietic stem cells (HSCs). Commitment to lymphoid, granulocytic/monocytic (GM), megakaryocytic and erythroid lineages occurs through the differentiation of immature progenitors, and is subject to spatial and temporal control by intrinsic and extrinsic factors [1,2]. High-resolution characterization of BM progenitor populations has been achieved and this critical advance has allowed detailed interrogation of the gene regulatory networks which govern normal homeostatic and malignant hematopoietic differentiation, both in clinical patient samples and adult transgenic mouse models [3,4,5,6,7,8,9,10,11,12,13,14,15,16]. The existence of an obligatory intermediate CMP population, as an ancestor of all committed granulocytic/monocytic, megakaryocytic and erythroid progenitors, is questioned [3,7]. Utilizing a Runx dual-reporter mouse model (P1-GFP::P2-hCD4) [17] (which reflects the alternate use of the two Runx promoters) we recently reported that the P2-hCD4- PreMegE fraction comprises pro-erythroid progenitors, whereas P2-hCD4+ PreMegEs are skewed in favor of megakaryocytic output [18]

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