Marking of definitive HSC precursors in E7.5-E8.5 embryos using an Abcg2-CreER lineage-tracing mouse model

Abstract

Abcg2, a member of the ATP-binding cassette transporter family, is expressed in adult hematopoietic stem cells (HSCs) and is required for the side population phenotype of adult bone marrow HSCs and other adult tissue-specific stem cells. Lineage tracing in adult mice using the Abcg2-Cre mouse model showed that Abcg2 marks HSCs, intestinal stem cells, and spermatogonial stem cells. It is unclear whether definitive HSCs or their precursors in early embryonic development can be marked by Abcg2 expression. Here, we treated pregnant Abcg2 Cre/Cre RosaLSL-YFP mice with a single injection of 4-hydroxytamoxifen at embryonic day 7.5. Four months after birth, a small yellow fluorescent protein-positive (YFP⁺) cell population could be detected in all of the major white blood cell lineages and this was stable for 8 months. Transplant of bone marrow cells or Sca1⁺YFP⁺ cells from these mice showed continued multilineage marking in recipient mice at 4 months. These results demonstrate that Abcg2 expression marks precursors to adult long-term repopulating HSCs at E7.5 to E8.5 and contributes to a stable subpopulation of HSCs well into adulthood.

Fig 1.. Abcg2 expression marks E7.5-E8.5 pdHSCs.

Pregnant Abcg2^CreERT2Rosa^EYFP mice were treated with a single injection of 4-OHT at E7.5. Fetuses were allowed to be born and grew to adult. Peripheral blood was taken at various time points and analyzed for YFP expression in the white blood cell lineages. Mice that had >1% YFP+ white blood cells at one month are graphed in A, and mice that had <1% YFP+ white blood cells at 1 month are graphed in B. YFP expression in white blood cell lineages from a representative mouse is shown in C. One Abcg2^CreERT2Rosa^EYFP mouse that did not receive 4-OHT showed absolutely no YFP expression (C, lower panels). Total bone marrow cells (D) or sorted Sca1+YFP+ cells (E) from mice treated with 4-OHT at E7.5 when 9 months old were transplanted into lethally irradiated recipient mice and YFP expression was analyzed in peripheral blood cell lineages 4 months after transplantation. YFP expression in lineages from one representative recipient mouse is shown in F.

Fig 1.. Abcg2 expression marks E7.5-E8.5 pdHSCs.

Pregnant Abcg2^CreERT2Rosa^EYFP mice were treated with a single injection of 4-OHT at E7.5. Fetuses were allowed to be born and grew to adult. Peripheral blood was taken at various time points and analyzed for YFP expression in the white blood cell lineages. Mice that had >1% YFP+ white blood cells at one month are graphed in A, and mice that had <1% YFP+ white blood cells at 1 month are graphed in B. YFP expression in white blood cell lineages from a representative mouse is shown in C. One Abcg2^CreERT2Rosa^EYFP mouse that did not receive 4-OHT showed absolutely no YFP expression (C, lower panels). Total bone marrow cells (D) or sorted Sca1+YFP+ cells (E) from mice treated with 4-OHT at E7.5 when 9 months old were transplanted into lethally irradiated recipient mice and YFP expression was analyzed in peripheral blood cell lineages 4 months after transplantation. YFP expression in lineages from one representative recipient mouse is shown in F.

Fig 2.. Low YFP marking in the blood allows estimation of the number of pdHSCs at E7.5-E8.5.

Abcg2^CreERT2Rosa^EYFP mice were treated with a single injection of 4-OHT at E7.5. Fetuses were allowed to be born and grew to adult. YFP expression in white blood cells at 8 months are shown for 8 mice.

Fig 3.. Lack of marking in kidney proximal tubules, hepatocytes and small intestine epithelial cells.

Abcg2^CreERT2Rosa^EYFP mice were treated with a single injection of 4-OHT at E7.5. Fetuses were allowed to be born and grew to adult and tissue sections were stained with anti-GFP antibody, anti Abcg2 antibody Bxp-53 and DAPI. Kidney (A), small intestine (B), liver (C) all at 20X magnification.