Fli-1 Transcriptionally Integrates Microenvironmental Cues Sensing by Self-Renewing Haematopoietic Stem and Progenitor Cells

INTRODUCTION

During adulthood and embryogenesis, fate decisions of haematopoietic stem and progenitor cells (HSPC), such as specification, self-renewal, and differentiation, are tightly regulated by their neighbouring niche cells.¹ Moreover, distinct types of niches supply differential cues to direct alternative cell fates for HSPC.^2,3 Currently however, the intrinsic mechanisms balancing HSPC response obliqueness to microenvironmental signals are unknown.

Fli-1 is an E26 transformation specific transcription factor expressed by vascular beds and haematopoietic lineages. Fli-1 belongs to the family of ‘heptad factors’ which are hypothesised to specify and sustain a haematopoietic cell fate.^4,5 While Fli-1 overexpression is linked to leukaemia,⁶ the functional role Fli-1 plays in HSPC specification and maintenance remains undefined.

METHOD AND RESULTS

The authors showed that inducible global deletion of Fli-1 using a Cre/lox model (Fli-1^ROSAΔ ) results in a rapid thrombocytopenia-associated mortality. Transplantation of Fli-1^ROSAΔ bone marrow cells into wild-type (WT) recipients, to exclude vascular-mediated defects, followed by induction of Fli-1 deletion resulted in the same phenotype. In a set of modulated competitive transplantation experiments (differential induction time points pre or post-transplant), the authors observed defective ability of Fli-1^ROSAΔ HSPC to lodge, engraft, and to sustain haematopoiesis post-repopulation. Fli-1-deficient HSPC exhibit reduced quiescence, a hallmark of stemness, and display enhanced apoptosis. Thus, Fli-1 is essential for previously unrecognised cell-autonomous HSPC functions.

To determine whether Fli-1 deletion abrogates haematopoietic specification, Fli-1 was conditionally deleted using a developmental haematopoietic Cre/lox model (Fli-1^Vav-1Δ) which resulted in premature mortality. Reduced presence of embryonic Fli-1^Vav-1Δ liver HSPC was observed at e12.5. Moreover, using a developmental endothelial (CDH5)-Cre/lox model (Fli-1^CDH5Δ), the authors observed that reduced numbers of haematopoietic cells were still detected in the aorta-gonad-mesonephros (AGM) region. Two in vitro co-culture systems were also applied to study Fli-1 in the endothelial-to-haematopoietic transition. First, isolated haemogenic endothelial cells from WT and Fli-1^ROSAΔ embryos were cocultured with AGM-derived vascular niche.⁷ Haemogenic endothelial cells isolated from Fli-1^ROSAΔ AGM were still able to convert to CD45+ cells, however these cells did not expand on a vascular niche. Secondly, an endothelial-to-haematopoietic reprogramming system was implemented in which isolated lung endothelial cells were virally introduced with DOX inducible FosB, Gfi1, Runx1, and Spi1 (FGRS) factors and co-cultured with vascular niche cells.^8,9 Both WT and Fli-1^ROSAΔ endothelial cells were able to acquire a haemogenic-like state resulting in a final capacity to convert into haematopoietic cells. Again, Fli-1^ROSAΔ cells displayed fewer numbers of CD45+ cells at the end point, presumably due to impaired interaction with the vascular niche. Induction of Fli-1 deletion in vitro in adult HSPC revealed loss of dependency on vascular niche inductive signals, as no additive expansion effect was observed for Fli-1^ROSAΔ HSPC in the presence of a vascular niche. Hence, Fli-1 is not essential for haematopoietic specification but rather essential for HSPC expansion and self-renewal.

Differential RNA-seq analysis combined with epigenetic studies of expanding WT and Fli-1^ROSAΔ HSPC revealed dysregulation of Fli-1-controlled pathways involved in transduction of microenvironmental signals for self-renewal. Focusing on HSPC-vascular niche interaction, this study observed dysregulation in Notch and VEGF pathway elements that mediate an HSPC-vascular niche crosstalk.¹⁰

CONCLUSION

Decrypting the mechanism(s) by which Fli-1 orchestrates HSPC self-renewal may promote an improved expansion protocol of human HSPC pre-transplantation and provide additional insights for microenvironmental sensing by Fli-1-dependent normal and leukaemic cells (Figure 1).