Thomas Clapes

GFI1 proteins orchestrate the emergence of haematopoietic stem cells through recruitment of LSD1

In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta–gonad–mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.

Ex vivo time-lapse confocal imaging of the mouse embryo aorta

Time-lapse confocal microscopy of mouse embryo slices was developed to access and image the living aorta. In this paper, we explain how to label all hematopoietic and endothelial cells inside the intact mouse aorta with fluorescent directly labeled antibodies. Then we describe the technique to cut nonfixed labeled embryos into thick slices that are further imaged by time-lapse confocal imaging. This approach allows direct observation of the dynamic cell behavior in the living aorta, which was previously inaccessible because of its location deep inside the opaque mouse embryo. In particular, this approach is sensitive enough to allow the experimenter to witness the transition from endothelial cells into hematopoietic stem/progenitor cells in the aorta, the first site of hematopoietic stem cell generation during development. The protocol can be applied to observe other embryonic sites throughout mouse development. A complete experiment requires ∼2 d of practical work.

Erythropoietic Defect Associated with Reduced Cell Proliferation in Mice Lacking the 26S Proteasome Shuttling Factor Rad23b

ABSTRACT Rad23a and Rad23b proteins are linked to nucleotide excision DNA repair (NER) via association with the DNA damage recognition protein xeroderma pigmentosum group C (XPC) are and known to be implicated in protein turnover by the 26S proteasome. Rad23b-null mice are NER proficient, likely due to the redundant function of the Rad23b paralogue, Rad23a. However, Rad23b-null midgestation embryos are anemic, and most embryos die before birth. Using an unbiased proteomics approach, we found that the majority of Rad23b-interacting partners are associated with the ubiquitin-proteasome system (UPS). We tested the requirement for Rad23b-dependent UPS activity in cellular proliferation and more specifically in the process of erythropoiesis. In cultured fibroblasts derived from embryos lacking Rad23b, proliferation rates were reduced. In fetal livers of Rad23b-null embryos, we observed reduced proliferation, accumulation of early erythroid progenitors, and a block during erythroid maturation. In primary wild-type (WT) erythroid cells, knockdown of Rad23b or chemical inhibition of the proteasome reduced survival and differentiation capability. Finally, the defects linked to Rad23b loss specifically affected fetal definitive erythropoiesis and stress erythropoiesis in adult mice. Together, these data indicate a previously unappreciated requirement for Rad23b and the UPS in regulation of proliferation in different cell types.

Embryonic development of hematopoietic stem cells: implications for clinical use

Hematopoietic stem cell (HSC) transplantation is an important treatment modality for hematological malignancies or to correct congenital immunodeficiency disorders. Several stem cell sources are currently applied clinically, with a recent increased application of umbilical cord blood. The low number of HSCs available, particularly in umbilical cord blood, is a limiting factor, and different lines of research are ongoing to circumvent this issue. In this review, we will describe the research strategies developed to expand adult HSCs in vitro and to generate new HSCs from pluripotent stem cell lines. We will also discuss the importance of studying the embryonic microenvironment since it allows both generation and extensive expansion of HSCs. Understanding the mechanisms that underlie HSC production, self-renewal and differentiation is necessary for the establishment of optimal in vitro HSC cultures, where a limitless and manipulatable resource of HSCs would be available for both clinical and fundamental research.