Hematopoietic Stem Cell Development During Mouse Embryogenesis

Julien Y. Bertrand, Sebastien Giroux, Ana Cumano, and Isabelle Godin

Summary

The progress of the last few years in the understanding of hematopoietic cell development during embryogenesis resulted from a combination of experimental approaches used in hematology and developmental biology. This methodology has been particularly powerful for the analysis of the earliest steps of hematopoietic ontogeny because it allows for the first time the demonstration of the existence of two independent sites of hematopoietic cell generation. Here, we describe the methods used in our laboratories to characterize the phenotype and differentiation potential of the primordial hematopoietic precursors as well as their localization in the mouse embryo. This multidisciplinary approach is required to explore the mechanisms of hematopoietic cell generation.

Key Words: AGM; flow cytometry; FTOC; in situ hybridization; hematopoiesis; mouse embryo; ontogeny; organ culture; stem cells; yolk sac.

1. Introduction

Hematopoietic progenitors are first detected in the yolk sac (YS) of mouse embryos between 7 and 7.5 d post-coitus (dpc) (1). These YS hematopoietic precursors can either be expanded in vitro or transplanted in vivo. Reconstitution of the hematopoietic lineage in recipient adult mice cannot be obtained under these conditions. Only eryth-roid cells are generated after in vitro expansion of YS hematopoietic progenitors under culture conditions that allow the differentiation of all hematopoietic lineages. Nowhere in the embryo proper can we find other hematopoietic cells at this stage of development (2,3).

Later, between 8 and 8.5 dpc, before the establishment of circulation between the YS and the embryo proper, hematopoietic precursors can also be detected in the intraembryonic compartment (splanchnopleura [Sp]: see following paragraph), but only if an organ culture step precedes in vitro differentiation or in vivo transplantation.

From: Methods in Molecular Medicine, Vol. 105: Developmental Hematopoiesis: Methods and Protocols. Edited by: M. H. Baron © Humana Press Inc., Totowa, NJ

Under these conditions, Sp precursors can generate all hematopoietic lineages when transferred to the appropriate differentiation culture systems and provide hematopoietic long-term reconstitution in natural killer-deficient mice, illustrating the presence of hematopoietic stem cells. In contrast, YS isolated from the same embryos generate only precursors with erythroid and/or myeloid potential and do not provide hematopoietic long-term reconstitution (3).

The extraembryonic and intraembryonic sites capable of generating hematopoietic cells before the onset of fetal liver hematopoiesis as well as their evolution during development are schematically described in Fig. 1. The extraembryonic YS provides for immediate erythropoiesis from 7 dpc. The hematopoietic determination of mesodermal cells takes place in the caudal intraembryonic Sp beginning at the presomitic stage (7.5 dpc). In both hemogenic sites, mesoderm is associated with endoderm, a combination termed Splanchnopleura. After the 15-somite stage, the tissues derived from the Sp comprise the endoderm of the developing gut, the dorsal aorta, the ompha-lomesenteric artery, and the splanchnopleural lining of these tissues. This site is now referred to as Para-aortic Sp (P-Sp: 8.5-10 dpc; ref. 4). When fetal liver colonization by hematopoietic stem cells begins, the P-Sp develops further and comprises, besides the aorta, the developing gonads and the mesonephros (AGM region: 10-11.5 dpc; refs. 5-8).

2. Materials

2.1. Dissection of Embryo

2. Phosphate-buffered saline (PBS) with calcium and magnesium.

3. Dissecting tools. Dissecting tools are all from BioTek Microsurgery, 34 Rue Des Chardonnerets, 92160 Antony, France; Web site: biotek-online.com:

a. Curved-serrated forceps: P-95-BC;

b. Fine forceps: DU-110-AUF

c. Ultra-fine forceps: DU-110-A

d. Sieve used for embryo transfer: S-20-P

2.2. Whole-Mount and In Situ Hybridization

1. Labeling solution: ribonucleic acid (RNA) labeling with digoxigenin-UTP by in vitro transcription with appropriate RNA polymerases (Boehringer), DIG RNA labeling. Mix (1X final), transcription buffer (1X final), RNA polymerase (40 ^L), RNAsine (10-20 ^.M/^L) and complemented with RNA free H2O to a 20 ^L of final volume.

2. TE: 10 mM Tris-HCl, 1 mM ethylenediamine tetraacetic acid (EDTA; pH 8.0), make up with RNAse-free water and autoclave

3. WISH-FIX: Whole-mount embryo in situ hybridization fixative solution (for 50 mL), 5 mL 37% formaldehyde, 200 ^L of 0.5 M EDTA (pH 8.8) and 150 ^L of 1 M NaOH in PBS without Ca2+/Mg2+.

4. WISH buffer: Whole-mount embryo in situ hybridization buffer (for 200 mL), 100 mL of formamide, 13 mL of 20X SSC (pH 5.0), 2 mL 0.5 M EDTA (pH 8.0), Torula (50 ^g/mL), 400 ^L of Tween-20, 1 g of CHAPS and heparin (100 ^g/mL) in H2O.

5. ISH-Fix buffer: in situ hybridization fixative buffer (for 200 mL): 8 g of sucrose, 24 ^L of 1 M CaCl2, 77 mL of 0.2 M NaHPO4, and 23 mL of 0.2 M NaH2PO4 in H2O.

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