In this paper, we examined the biological role of Zfx
, one of the first identified zinc finger-encoding genes (Schneider-Gadicke et al., 1989
) whose properties and function remained unclear. We found that Zfx deletion impaired the growth of ESC in vitro
and increased their apoptosis rate, particularly in defined serum-free conditions. Despite the growth and survival defect, Zfx-deficient ESC remained undifferentiated and gave rise to all lineages, including the germ line. Conversely, stable Zfx overexpression favored ESC self-renewal at the expense of differentiation. The requirement for Zfx in ESC self-renewal in vitro
is not contradicted by the relatively normal early development of Zfxnull
embryos, which survive until mid-gestation. Indeed, early embryonic development is not affected by the loss of several ESC self-renewal regulators such as Eras (Takahashi et al., 2003
), Tcl1, Essrb or Tbx3 (Ivanova et al., 2006
). Thus, our data suggest a critical role of Zfx in promoting ESC self-renewal, while it appears dispensable for ESC pluripotency.
In addition, we found that Zfx is not required for fetal hematopoiesis but essential in adult HSC, which failed to maintain their numbers or contribute to hematopoiesis after Zfx deletion. Zfx-deficient HSC interacted normally with their BM niche but showed an increase in apoptosis, suggesting a defect of long-term self-renewal due to impaired survival. In contrast, the survival and function of erythromyeloid progenitors were not affected, at least in the short term. Very similar phenotypes have been observed after the inducible deletion of Tel/Etv6 (Hock et al., 2004
) or FoxO1/3/4 (Tothova et al., 2007
), or after germ line deletion of Bmi-1 (Park et al., 2003
) or Ikaros (Nichogiannopoulou et al., 1999
) transcription factors. Similar to the latter three models, lymphopoiesis was severely impaired after the loss of Zfx. Thus, Zfx and possibly other HSC regulators appear to control both HSC self-renewal and lymphoid differentiation, but not erythromyeloid differentiation. Alternatively, the failure of lymphoid development might be secondary to defective HSC function, while erythromyeloid progenitors might be less dependent on HSC input (Nichogiannopoulou et al., 1999
Although several transcriptional regulators were shown to control multiple SC types, the common regulation of ESC and adult SC has been difficult to demonstrate. For instance, Polycomb group
(PcG) protein Bmi-1 promotes the self-renewal of HSC and of neural SC through the repression of Cdkn2a
(Lessard and Sauvageau, 2003
; Molofsky et al., 2003
; Park et al., 2003
). On the other hand, ESC are resistant to p16ink4a
and to other cyclin inhibitors (Burdon et al., 2002
), and are not known to depend on Bmi-1. Conversely, PcG protein Eed contributes to the repression of differentiation-associated genes in ESC (Azuara et al., 2006
; Boyer et al., 2006
); however, Eed promotes proliferation and antagonizes Bmi-1 in hematopoietic cells (Lessard et al., 1999
). In another case, transcription factor Sox2 controls the pluripotency of ESC and of neural stem/progenitor cells (Boyer et al., 2005
; Graham et al., 2003
; Ivanova et al., 2006
). However, Sox2 is not expressed in other adult SC types, including HSC, and its specific role in adult neural SC remains to be established. Thus, Zfx provides a rare example of a transcription factor that is specifically required in ESC and in a canonical adult SC population such as the HSC.
Because the common transcriptional regulation of ESC and HSC has not been previously described, it is likely to be based on equally novel molecular mechanisms. Indeed, we have identified several direct target genes of Zfx in ESC and HSC, all of which are either predicted or poorly characterized. While the exact function of these direct Zfx targets is currently unknown, their coordinate regulation in ESC and HSC supports the common role of Zfx in these SC types. In addition, we identified several genes upregulated in Zfx-deficient ESC and HSC, likely as an indirect consequence of Zfx loss. Most of the coordinately induced genes are targets of stress, growth factor signaling and/or immediate-early response, suggesting that Zfx-deficient SC undergo excessive stress. This is consistent with increased apoptosis rate of Zfxnull
ESC and HSC; furthermore, both immediate-early gene induction and decreased survival occur in undifferentiated ESC and HSC, but not in their differentiated progeny. Of note, HSC are uniquely sensitive to certain forms of stress such as physiological oxidative stress (Tothova et al., 2007
). Altogether, our data reveal the apoptosis and concomitant upregulation of stress-inducible genes as common features of impaired self-renewal in ESC and HSC.
In addition to the common target genes, Zfx can directly activate cell type-specific targets, such as Tbx3
in ESC. Both genes are rapidly downregulated upon ESC differentiation, and facilitate ESC self-renewal through a putative common pathway (Ivanova et al., 2006
). Recently, Tcl1 was shown to control ESC growth but not their differentiation status (Matoba et al., 2006
). Thus, Zfx may promote ESC self-renewal at least in part through the direct coordinate regulation of Tbx3 and Tcl1. Furthermore, the ESC-specific expression of Tbx3 and Tcl1 may partially explain the requirement for Zfx in ESC but not in their differentiated progeny. Although none of the known HSC regulators were affected by the loss of Zfx (Suppl. Table III
and data not shown), Zfx may similarly regulate a yet unknown HSC-specific self-renewal pathway. At present, the ESC system provides a proof of principle that Zfx can control self-renewal pathways specific for a particular SC type.
In conclusion, our study has identified Zfx as a shared transcriptional regulator of two prototypical yet highly different murine SC types, the embryonic and hematopoietic SC. These results provide genetic evidence for a common molecular basis of self-renewal in pluripotent embryonic SC and adult tissue-specific SC. The identification of Zfx should facilitate the molecular dissection of self-renewal mechanisms in ESC, HSC and possibly in other SC types, including tumor-initiating cancer SC. Finally, the high evolutionary conservation of Zfx raises the possibility that it may regulate SC functions in other vertebrate species, including humans.