All primitive and definitive hematopoietic progenitor cells emerging before E10 in the mouse embryo are products of the yolk sac

Abstract

The relative contribution of yolk sac (YS)-derived cells to the circulating definitive hematopoietic progenitor cell (HPC) pool that seeds the fetal liver remains controversial due to the presence of systemic circulation and the onset of hematopoiesis within the embryo proper (EP) before liver seeding. Ncx1-/- embryos fail to initiate a heartbeat on embryonic day (E) 8.25, but continue to develop through E10. We detected normal numbers of primitive erythroid progenitors in Ncx1-/- versus wild type (WT) YS, but primitive erythroblasts did not circulate in the Ncx1-/- EP. While there was no significant difference in the number of definitive HPCs in Ncx1-/- versus WT YS through E9.5, the Ncx1-/- EP was nearly devoid of HPCs. Thus, primitive erythroblasts and essentially all definitive HPCs destined to initially seed the fetal liver after E9.5 are generated in the YS between E7.0-E9.5 and are redistributed into the EP via the systemic circulation.

Figure 1

The Ncx1^−/− mouse embryo provides an in vivo circulation-free environment. Ncx1 mutants were generated via insertion of LacZ reporter into exon 2 of the Ncx1 gene, thus all cells that normally express Ncx1 can be labeled with X-gal staining. (A) E9.5 embryos (WT(i), Ncx1^+/−(ii), and Ncx1^−/−(iii)) demonstrate that development continues well past the onset of circulation. (B) X-gal staining (∼27 sp) reveals that expression of Ncx1 is restricted to the heart through E10. Importantly, there is no expression in any putative site of hematopoietic development including the YS and PSp region nor in the hematopoietic cells themselves (see blood vessels in Ncx1^+/− YS). (C) Ncx1 RT-PCR was conducted on embryonic tissues from various ages to confirm the X-gal staining. At E8.0, Ncx1 is not yet detectable in either the embryo proper or YS. At E9.0 and E11.5, Ncx1 is detected exclusively in the heart cardiomyocytes. (D) Ten-micrometer sagittal sections of hematoxylin and eosin (H&E) stained E9.25 (19 sp) embryos. Panels Di,iii are 100× magnifications of sections that best profile the structure of the PSp region (*), which is clearly present in both WT and Ncx1^−/− embryos. Image Di was cut at an oblique angle compared with image Diii, leaving only a small portion of the heart and upper body in view. Image Dv is an insert that transects the hypoplastic Ncx1^−/− heart. Images Dii,iv are 200× magnifications of the PSp (*) regions. Endothelial cells can be seen lining the vessels (below yellow line) and circulating blood cells (arrows) are seen in the WT embryo but are notably absent in the Ncx1^−/− embryo. The images in panels A and B were viewed on a Leica MZ9.5 Stereomicroscope (1.0× Planachromatic Lens/0.20 NA)(IL-3) with DFC320 CCD camera, captured with Leica application suite (LAS) software (Leica, Bannockburn, IL). Original magnification, ×60. The images in panel D were viewed on a Zeiss Axioskop Stereomicroscope (Zeiss Plan-Neofluor 10×/0.30 NA (top) and 20×/0.50 NA (bottom)) with SPOT RTKE cooled color CCD camera, and imported into the SPOT Advanced software (Diagnostic Instruments, Sterling Heights, NJ). Original magnification, ×100.

Figure 2

Circulation is required to distribute hematopoietic progenitors and differentiated cells to the embryo proper. (A) Primitive progenitor colony number is not altered by the lack of flow in Ncx1^−/− embryos. WT embryos produced an average of 1617 (± 255; mean ± SEM) colonies while Ncx1^−/− embryos produced 1697 (± 227) colonies. (B) ζ-globin in situ hybridization specifically labels the primitive erythroblast lineage. At 6 sp, just after the first heart beats, no difference in blood distribution is detectable in Ncx1^−/− or WT embryos. Note the cluster of cells in the embryo proper at the site of connection of the vitelline vasculature (*). At 14 sp, a significant amount of ζ-globin staining is detected in the embryo proper of the WT while the Ncx1^−/− embryo remains devoid of erythroblasts. (C) Embryos (23 sp) were stained with benzidine, which stains all hemoglobin-containing cells dark blue/black. At this stage of development, all hemoglobin-containing erythroblasts are derived from primitive progenitors from the yolk sac (YS) and the vast majority of staining occurs in the YS. Note Ncx1^−/− EP has no stained erythroblasts compared with age-matched WT littermates. (D) (Representative definitive progenitor numbers) There is an expansion of definitive HPCs in both the YS and embryo proper from E8.5 of development through E9.5 in the WT embryos (Table S1). Definitive HPCs in the YS expand from 82 to 578 (colonies/YS) and in the PSp from 5 to 115 (colonies/PSp) during this time period. In the absence of a circulation in the Ncx1^−/− embryo, definitive HPC numbers in the YS range from 79 to 559 and are not significantly different from WT littermates. The Ncx1^−/− PSp ranges from 0 to 3 definitive HPCs, which is a significant decrease (P < .05 by paired Student t test) from WT at all timepoints examined. Images in panels B,C acquired as for Figures 1A and B. Original magnification, ×60.