Given the importance of proliferation in cell commitment and

Given the importance of proliferation in cell commitment and differentiation, here we have studied proliferative changes during HSC maturation steps, which to date have not been studied in detail. We showed previously that in culture developing HSCs of the AGM region proliferate slower than committed progenitors (Taoudi et al., 2008). More recent in vivo analysis of the dramatic pre-HSC expansion in the AGM region suggests that proliferation or/and cell recruitment may play a role (Rybtsov et al., 2016). In vitro modeling has proved to be a powerful and informative approach for the identification of pre-HSC states and dissection of HSC developmental mechanisms (Taoudi et al., 2008). HSCs develop through a multi-step process: pro-HSC → pre-HSC I → pre-HSC II → dHSC, which involves sequential upregulation of hematopoietic markers CD41 (Itga2b), RUNX1 (AML1), CD43 (Spn), and CD45 (Ptprc) in VE-CADHERIN+ (VC) precursors (Rybtsov et al., 2011, 2014; Taoudi et al., 2008; Medvinsky and Dzierzak, 1996; Liakhovitskaia et al., 2014; Swiers et al., 2013; Yoder et al., 1997). Pro-HSCs (VC+CD41loCD43−CD45−) emerge at embryonic day 9.5 (E9.5), pre-HSCs type I (VC+CD41loCD43+CD45−) at E10.5, and pre-HSCs type II (VC+CD41loCD43+CD45+) at E11.5 stages. Low dHSC numbers emerge at E11.5 and, although phenotypically similar to pre-HSCs type II, they can be detected by direct transplantations into irradiated recipients. Pro-/pre-HSCs have been identified in hematopoietic clusters budding from the endothelium of major embryonic lp-pla2 (Rybtsov et al., 2011, 2014; Taoudi et al., 2008; Yokomizo and Dzierzak, 2010; Kissa and Herbomel, 2010; Boisset et al., 2011; Gordon-Keylock et al., 2013; Ciau-Uitz et al., 2016). Functional assessment of cell proliferation in live cells often involves Hoechst staining, which can be toxic and can alter the experimental outcome (Parish, 1999). Instead, we used the fluorescent ubiquitination-based reporter (Fucci) system, which enables noninvasive in vivo visualization of the cell-cycle status and their isolation for functional analysis (Sakaue-Sawano et al., 2008; Yo et al., 2015; Zielke and Edgar, 2015). We describe here that pro-HSCs (at E9.5) initially slowly cycle, then enter active proliferation during E10.5–E11.5, which correlates with the expansion of the pro-/pre-HSC pool (Rybtsov et al., 2016). However, this phase is followed by gradual slowing down of proliferation, the first signs of which can be already observed in AGM dHSCs, in keeping with gradual acquisition of adult status by dHSCs. We also describe the orderly architectural evolvement of intra-aortic clusters in which stepwise HSC maturation and proliferation are linked. It is suggested that the proliferative pattern within the cluster is defined by c-KIT/SCF signaling.
Results
Discussion Cell proliferation plays an important role in various developmental processes. It underlies growth of tissues and organs and is involved in cell-fate decisions (Fuchs, 2009; Pauklin and Vallier, 2013). Proliferation is an important mechanism enabling self-renewal and differentiation of HSCs in the adult (Bowie et al., 2006; Pietras et al., 2011). Here we used Fucci reporter mice to define the proliferative status of the developing HSCs. Our conclusions based on the ratio of Geminin-mAG+ and Cdt1-mKO2+ cells are consistent with previously described proliferation rates of fetal liver and bone marrow HSCs (Bowie et al., 2006, 2007a; Nygren et al., 2006, 2007b; Takizawa et al., 2011; Fuchs, 2009). During maturation, the developing HSC pool undergoes massive expansion within the AGM region before colonization of the fetal liver (Rybtsov et al., 2016). HSC maturation occurs through sequential upregulation of hematopoietic markers (CD41, CD43, and CD45) (Taoudi et al., 2008; Rybtsov et al., 2014). Fucci mice enabled visualization and isolation of cells in G0/G1 (red) and S/G2/M (green) phases, so that developing HSCs could be studied at both the phenotypic and functional levels (Figure 5A). We found that the Geminin-mAG− but not Geminin-mAG+ fraction of the E9.5 pro-HSC population was able to mature into dHSCs, which could reconstitute adult irradiated recipients, indicating that pro-HSCs are not cycling or slowly cycling. By the next day (E10.5), upregulation of CD43 marks the emergence of pre-HSCs type I, which are actively proliferating since both Geminin-mAG+ and Geminin-mAG− fractions can produce dHSCs. Thus, proliferation likely underlies the previously described dramatic expansion of the pre-HSC pool (from 5 cells at early E10 to 50 cells by late E10.5) (Rybtsov et al., 2016). At E11.5 pre-HSCs type II mature and continue to proliferate, with some bias toward the Geminin-mAG− fraction, indicating a slowing down in this process, which becomes apparent in dHSCs. This slowing down of the cell cycle continues further in fetal liver HSCs and, finally, in mainly quiescent bone marrow HSCs (Figure 5A) (Bowie et al., 2007a; Yo et al., 2015).