Abstract

Notch is essential for the generation of definitive hematopoietic stem cells (HSCs) during fetal life, but its effects on subsequent HSC self-renewal are controversial. We have shown recently that canonical Notch signaling is dispensable for the maintenance of stem cell function in adult HSCs (Maillard et al., submitted). We now investigate whether Notch regulates HSC homeostasis during fetal development. For this purpose, we have developed a system to track individual Notch-deprived cells through expression of the GFP-tagged pan-Notch inhibitor DNMAML (Maillard et al., Blood 2004). We have used this system to conditionally inactivate Notch signaling at defined time points during development. Specifically, we crossed mice carrying an ubiquitously expressed Tamoxifen(TAM)-inducible Cre recombinase transgene (CAGG-CreERTm) with mice harbouring a Cre-inducible DNMAML construct (DNMAMLf). We injected TAM i.p. at mid-gestation (E11.5), after generation of fetal HSCs. At E13.5, DNMAML-GFP protein expression was induced in 20–45% of hematopoietic progenitors in the Lin-Sca-1hic-Kithi (LSK) fraction. As compared to control littermates, TAM-induced CAGG-CreERTm x DNMAMLf (TICD) fetuses initially showed increased numbers and decreased quiescence of fetal liver LSK cells: at E13.5, TICD LSK progenitors had increased cell size, expressed elevated levels of Sca-1, and showed an increase in the fraction of cells in S-G2M phases of the cell cycle. Significantly, these abnormalities were observed both within the GFP- and GFP+ TICD LSK fractions, indicating non-cell-autonomous disruption of their homeostasis. At subsequent time points (E15.5), TICD fetuses showed a marked decrease in the number of LSK progenitors, most prominently within the CD150+CD48− subset highly enriched for long-term HSCs (LT-HSC), suggesting HSC exhaustion. Histological examination revealed multiple small areas of hemorrhage in TICD tissues consistent with underlying vascular defects. Fetal death was observed at E17.5–18.5. Altogether, our findings show that continuous Notch signaling is absolutely required for normal development during mid-gestation and suggest that Notch controls a key element of the fetal HSC niche that promotes LT-HSC self-renewal and quiescence. Because inactivation of Notch signaling causes phenotypic changes associated with vascular defects, we speculate that Notch regulates critical vascular elements of the stem cell niche in the fetal liver.

Disclosures: Damon Runyon Cancer Research Foundation, NIH.

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