Retinoic acid orchestrates fibroblast growth factor signalling to drive embryonic stem cell differentiation

INTRODUCTION Differentiating mouse embryonic stem (ES) cells first downregulate pluripotency genes and transit through a primitive ectoderm-like state before adopting somatic cell fates. Blocking fibroblast growth factor (Fgf) signalling via the Erk1/2 MAP kinase (Mapk) pathway promotes self-renewal in mouse ES (mES) cells (Burdon et al., 1999; Ying et al., 2008) (reviewed by Silva and Smith, 2008), whereas exposure to such signalling is required for differentiation into neural and non-neural cell fates (Kunath et al., 2007; Stavridis et al., 2007). A period of Fgf/Erk signalling thus appears to be a first step away from the self-renewal cell state in this context. It is also evident, at least for neural differentiation, that such signals are only briefly required because, after 24 hours, blocking Erk signalling no longer inhibits the later onset of the neural progenitor marker Sox1 (Stavridis et al., 2007). This suggests that a particular Fgf/Erk signalling dynamic could underlie differentiation progression. Another signalling molecule, retinoic acid (RA), also promotes differentiation in a range of tissues and cell lines, as well as in ES cells. Depending on culture conditions, retinoid signalling can promote ES cell differentiation into endoderm, adipocytes, fibroblast-like cells or neural tissue (reviewed by Soprano et al., 2007). In particular, neural fate is routinely elicited from ES cell aggregates (embryoid bodies) cultured in differentiation conditions for 4 days and then treated with RA (e.g. Aubert et al., 2002; Bain et al., 1995). The inclusion of a long period in an aggregated form in this protocol prior to exposure to RA suggests that, in this cellular context, RA acts after loss of self-renewal to promote differentiation. However, there is also evidence that RA has an earlier action via repression of the pluripotency gene Oct4 in ES cells (Gu et al., 2005) and that it might work directly via a retinoic acid response element (RARE) in the Oct4 promoter, as has been reported in P19 EC cells (Barnea and Bergman, 2000; Okazawa et al., 1991; Pikarsky et al., 1994). This may be one way in which RA influences Fgf signalling during differentiation, as Oct4 together with Sox2 promotes Fgf4 expression in ES cells (Yuan et al., 1995). Fgf4 is the principal source of Erk signalling in differentiating mES cells, as indicated by reduced dP-Erk levels and the poor differentiation of Fgf4–/– ES cells (Kunath et al., 2007), and so RA could limit an initial period of high Fgf/Erk activity by repressing Oct4. The regulatory relationship between retinoid and Fgf signalling pathways differs depending on the cellular context. In the mouse and chick embryonic axis, RA promotes differentiation by inhibiting expression of Fgf8 as cells leave the tailbud (reviewed by Wilson et al., 2009); a step that may be analogous to RA-mediated downregulation of Fgf4 in ES cells. Furthermore, RA and Fgf pathways are mutually inhibitory in the embryonic axis (Diez del Corral et al., 2003), and elevated Fgf or reduced RA signalling is characteristic of many cancerous cell states (reviewed by Diez del Corral and Storey, 2004). These studies therefore suggest that RA attenuation of Fgf signalling is a fundamental signalling mechanism controlling cellular differentiation. In other contexts, however, RA can promote Fgf8 transcription, for example, in neurula-stage frog embryos (Moreno and Kintner, 2004). There is also evidence that RA receptors (RARs) can bind Fgf8 upstream elements (Brondani et al., 2002; Zhao et al., 2009), indicating that RA might directly regulate Fgf8. In addition, RA can activate the Erk pathway by so-called nonDevelopment 137, 881-890 (2010) doi:10.1242/dev.043117 © 2010. Published by The Company of Biologists Ltd