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Concealed expansion of immature precursors underpins acute burst of adult HSC activity in foetal liver

Rybtsov, Stanislav, Ivanovs, Andrejs, Zhao, Suling, Medvinsky, Alexander·2016·Development
DOI 10.1242/dev.131193PMC PMC4852516
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Abstract

One day prior to mass emergence of haematopoietic stem cells (HSCs) in the foetal liver at E12.5, the embryo contains only a few definitive HSCs. It is thought that the burst of HSC activity in the foetal liver is underpinned by rapid maturation of immature embryonic precursors of definitive HSCs, termed pre-HSCs. However, because pre-HSCs are not detectable by direct transplantations into adult irradiated recipients, the size and growth of this population, which represents the embryonic rudiment of the adult haematopoietic system, remains uncertain. Using a novel quantitative assay, we demonstrate that from E9.5 the pre-HSC pool undergoes dramatic growth in the aorta-gonad-mesonephros region and by E11.5 reaches the size that matches the number of definitive HSCs in the E12.5 foetal liver. Thus, this study provides for the first time a quantitative basis for our understanding of how the large population of definitive HSCs emerges in the foetal liver.

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Fig. 1. Comparative limiting dilution analysis of pre-HSC sites. (A) Standard limiting dilution analysis for dHSCs using direct transplantations of freshly isolated cells. (B) Pre-HSC limiting dilution assay (preHSC-LDA). AGM cell suspension contains pro-HSCs and Type I pre-HSCs (green circles), Type II pre-HSCs (orange circles) and dHSCs (red circles). Each whole co-aggregate was injected in an individual recipient (one co-aggregate per mouse). Note that 0.01 ee dose for co-aggregation is indicated as an example and varied in actual experiments. For further description, see Materials and Methods. (C) Dissection boundaries (green dashed lines) of cultured and transplanted tissues (E11.5). Position of the dorsal aorta (including bifurcated rostral part) is shown by thick orange line. (D) Tissue distribution of pre-HSCs. Each isolated tissue (as indicated in C) was co-aggregated individually with OP9 cells, cultured and each co-aggregate transplanted individually into a recipient (1 ee/co-aggregate/recipient). Blue diamonds represent donor-derived blood chimerism (%) in individual mice. Numbers of non-repopulated mice were used for quantification of pre-HSC as described in Materials and Methods for preHSC_LDA. AGM, aorta-gonad-mesonephros; BA, bifurcated dorsal aorta; BW, body wall; EEV, extra-embryonic vessels; FL, foetal liver; H, head; Pl, placenta; S, somites; T, tail; YS, yolk sac.
Fig. 1. Comparative limiting dilution analysis of pre-HSC sites. (A) Standard limiting dilution analysis for dHSCs using direct transplantations of freshly isolated cells. (B) Pre-HSC limiting dilution assay (preHSC-LDA). AGM cell suspension contains pro-HSCs and Type I pre-HSCs (green circles), Type II pre-HSCs (orange circles) and dHSCs (red circles). Each whole co-aggregate was injected in an individual recipient (one co-aggregate per mouse). Note that 0.01 ee dose for co-aggregation is indicated as an example and varied in actual experiments. For further description, see Materials and Methods. (C) Dissection boundaries (green dashed lines) of cultured and transplanted tissues (E11.5). Position of the dorsal aorta (including bifurcated rostral part) is shown by thick orange line. (D) Tissue distribution of pre-HSCs. Each isolated tissue (as indicated in C) was co-aggregated individually with OP9 cells, cultured and each co-aggregate transplanted individually into a recipient (1 ee/co-aggregate/recipient). Blue diamonds represent donor-derived blood chimerism (%) in individual mice. Numbers of non-repopulated mice were used for quantification of pre-HSC as described in Materials and Methods for preHSC_LDA. AGM, aorta-gonad-mesonephros; BA, bifurcated dorsal aorta; BW, body wall; EEV, extra-embryonic vessels; FL, foetal liver; H, head; Pl, placenta; S, somites; T, tail; YS, yolk sac.CC BY · PubMed Central
Fig. 2. Quantitative developmental dynamics of pre-HSCs and HSCs in early embryo. (A-C) Dynamics of pre-HSC (green) and dHSC (red) numbers in caudal part/AGM region (A), extra-embryonic vessels (B) and foetal liver (C). Predicted polynomial regression curve, equation and correlation (R2) for pre-HSCs (green) and dHSCs (red) for each tissue are shown. Days post-fertilisation are indicated along the top and somite counts are along the lower axis. Each symbol represents a pre-HSC/dHSC number obtained from an individual experiment. Note that each symbol represents pre-HSC or HSC number in an individual experiment (not in an individual animal) calculated by preHSC_LDA (in green) or dHSC LDA (in red; as assayed by direct transplantations of fresh tissues); for methodology see Fig. 1B and 1A, respectively. For example, the curve for pre-HSCs in the AGM region represents 27 experiments. Full summary of experiments with repopulation levels of individual mice is presented in Table S2. Note that green dashed line (marked by asterisk) (C), represents pre-existing dHSCs in the foetal liver, which are maintained in culture, as numbers coincide with those calculated by direct transplantations shown in red (for detailed explanation, see Results).
Fig. 2. Quantitative developmental dynamics of pre-HSCs and HSCs in early embryo. (A-C) Dynamics of pre-HSC (green) and dHSC (red) numbers in caudal part/AGM region (A), extra-embryonic vessels (B) and foetal liver (C). Predicted polynomial regression curve, equation and correlation (R2) for pre-HSCs (green) and dHSCs (red) for each tissue are shown. Days post-fertilisation are indicated along the top and somite counts are along the lower axis. Each symbol represents a pre-HSC/dHSC number obtained from an individual experiment. Note that each symbol represents pre-HSC or HSC number in an individual experiment (not in an individual animal) calculated by preHSC_LDA (in green) or dHSC LDA (in red; as assayed by direct transplantations of fresh tissues); for methodology see Fig. 1B and 1A, respectively. For example, the curve for pre-HSCs in the AGM region represents 27 experiments. Full summary of experiments with repopulation levels of individual mice is presented in Table S2. Note that green dashed line (marked by asterisk) (C), represents pre-existing dHSCs in the foetal liver, which are maintained in culture, as numbers coincide with those calculated by direct transplantations shown in red (for detailed explanation, see Results).CC BY · PubMed Central
Fig. 3. Dynamic composition of pre-HSC/dHSC development. (A) Growing pool of pre-HSCs in the AGM region builds up by E11.5 and rapidly colonises the foetal liver (FL) by E12.5 (during 12-24 h) (cells are colour coded as in Fig. 1). Ao, dorsal aorta. (B) Cumulative number of pro-HSCs and Type I pre-HSCs (green area) relative to more mature Type II pre-HSCs (orange) and dHSCs (pink), which appear and expand at later stages in the AGM region. Numbers of AGM-derived pre-HSCs match dHSC numbers in E12 foetal liver (brown). Foetal liver is represented by brown area; the AGM region is represented by green, orange and pink areas. Each experimental point is the mean of three or four experiments (standard deviations are shown) and derived from Table S2 (calculated using preHSC_LDA). Ratio between pre-HSC Type I and II derived from Table S1 and Fig. S1B,C.
Fig. 3. Dynamic composition of pre-HSC/dHSC development. (A) Growing pool of pre-HSCs in the AGM region builds up by E11.5 and rapidly colonises the foetal liver (FL) by E12.5 (during 12-24 h) (cells are colour coded as in Fig. 1). Ao, dorsal aorta. (B) Cumulative number of pro-HSCs and Type I pre-HSCs (green area) relative to more mature Type II pre-HSCs (orange) and dHSCs (pink), which appear and expand at later stages in the AGM region. Numbers of AGM-derived pre-HSCs match dHSC numbers in E12 foetal liver (brown). Foetal liver is represented by brown area; the AGM region is represented by green, orange and pink areas. Each experimental point is the mean of three or four experiments (standard deviations are shown) and derived from Table S2 (calculated using preHSC_LDA). Ratio between pre-HSC Type I and II derived from Table S1 and Fig. S1B,C.CC BY · PubMed Central

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Citations

Cites

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Metadata

Authors
Rybtsov, S., Ivanovs, A., Zhao, S., & Medvinsky, A.
Venue
Development
Publisher
The Company of Biologists
Published
2016-04-15
Volume
143
Issue
8
Pages
1284-1289
ISSN
  • 1477-9129
  • 0950-1991
DOI
10.1242/dev.131193
PMC
PMC4852516
Cited by
136
References
2
Language
en
Licence
creativecommons.org/licenses/by/3.0 (vor, 1604 d)
Funders
  • Biotechnology and Biological Sciences Research Council
  • Leukaemia and Lymphoma Research
  • Medical Research Council
  • Wellcome Trust

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