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Review
. 2010;54(6-7):1003-9.
doi: 10.1387/ijdb.103118cl.

Novel methods for determining hematopoietic stem and progenitor cell emergence in the murine yolk sac

Christopher T Lux  1 , Mervin C Yoder
Affiliations
Review

Novel methods for determining hematopoietic stem and progenitor cell emergence in the murine yolk sac

Christopher T Lux et al. Int J Dev Biol. 2010.

Abstract

The mammalian yolk sac is known to play a prominent role in emergence of the hematopoietic system. The extent of this contribution has been a subject of debate in recent years largely due to effects of the early circulation that obscures the site of origin of hematopoietic stem and progenitor cells. This review discusses the limitations of some of the standard assays currently employed to study hematopoietic stem and progenitor cell emergence and highlights several recently reported novel methods that address this problem from new perspectives. Two methods directly alter the circulation by either preventing it from occurring in the first place or by removing vascular connections between the embryo and the yolk sac. Other approaches have altered the ability of hematopoietic cells to interact with their environment, resulting in the lack of migration or an inability to bind to potential hematopoietic niches. A third set of experiments utilize lineage tracing techniques to follow the migration of early progenitors once they enter the circulation. Taken together, these novel methods provide new evidence for the contribution of yolk sac hematopoietic stem and progenitor cells to the adult hematopoietic system.

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Figures

Fig. 1
Fig. 1. The Ncx1 null mutant mouse embryo provides an in vivo circulation-free environment for study of blood cell emergence
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 100x 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 200x 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 (1.0xPlanachromatic Lens/0.20 NA)(IL-3) with DFC320 CCD camera, captured with Leica application suite (LAS) software (Leica, Bannockburn, IL). Original magnification, x60. The images in panel D were viewed on a Zeiss Axioskop Stereomicroscope (Zeiss Plan-Neofluor 10x/0.30 NA (top) and 20x/0.50 NA (bottom)) with SPOT RTKE cooled color CCD camera, and imported into the SPOTAdvanced software (Diagnostic Instruments, Sterling Heights, NJ). Original magnification, x100. Reproduced without modification and with permission from Lux CJ et al. Blood, 2008: 111: 3435–3438.
Fig. 2
Fig. 2. Phenotypic analysis of wild type and Rac1 mutant embryos at E11.5
(A,B) Gross appearance of surviving TGRac1Flox/null″ embryos and littermate controls at E11.5. The limbs from the right side of the embryos were removed for clarity of presentation. The appearance of the littermate controls was similar for NTGRac1Flox/Wt, NTGRac1Flox/null, and TGRac1Flox/Wt (hereafter referred to as “control”). The anatomic region of the fetal liver is delineated and noted by arrows. The arrowheads point to accumulation of blood in cephalic regions as well as areas underlying the spinal cord, a phenotype previously noted in mutant animals deficient in hematopoietic cell production at the fetal liver stage. (C) Quantitative analysis of fetal liver hematopoiesis as analyzed by colony forming units/fetal liver of F2 embryos at E11.5. Data represent mean (±SD) of at least 3 embryos analyzed for each genotype. *P<.01, TGRac1Flox/null versus all other genotypes. Reproduced without modification and with permission from Ghiaur G et al. Blood, 2008: 111: 3313–3321.
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