Unexpected activities of Smad7 in Xenopus mesodermal and neural induction

Abstract

Neural induction is widely believed to be a direct consequence of inhibition of BMP pathways. Because of conflicting results and interpretations, we have re-examined this issue in Xenopus and chick embryos using the powerful and general TGFbeta inhibitor, Smad7, which inhibits both Smad1- (BMP) and Smad2- (Nodal/Activin) mediated pathways. We confirm that Smad7 efficiently inhibits phosphorylation of Smad1 and Smad2. Surprisingly, however, over-expression of Smad7 in Xenopus ventral epidermis induces expression of the dorsal mesodermal markers Chordin and Brachyury. Neural markers are induced, but in a non-cell-autonomous manner and only when Chordin and Brachyury are also induced. Simultaneous inhibition of Smad1 and Smad2 by different approaches does not account for all Smad7 effects, indicating that Smad7 has activities other than inhibition of the TGFbeta pathway. We provide evidence that these effects are independent of Wnt, FGF, Hedgehog and retinoid signalling. We also show that these effects are due to elements outside of the MH2 domain of Smad7. Together, these results indicate that BMP inhibition is not sufficient for neural induction even when Nodal/Activin is also blocked, and that Smad7 activity is considerably more complex than had previously been assumed. We suggest that experiments relying on Smad7 as an inhibitor of TGFbeta-pathways should be interpreted with considerable caution.

Calreticulin is a secreted BMP antagonist, expressed in Hensen's node during neural induction

“…The coding regions of chick Calreticulin (CALR), zebrafish Calreticulin (calr) (Rubinstein et al, 2000), human Calnexin (CANX) (kind gift from Marek Michalak (Vassilakos et al, 1998), Xenopus truncated BMP receptor (Suzuki et al, 1994), cSmad6 (a kind gift from P Szendro and G Eichele) (de Almeida et al, 2008, Yamada et al, 1999), cChordin (Streit et al, 1998) and xSmad7 (Casellas and Brivanlou, 1998, de Almeida et al, 2008) were each cloned into pCAβ-IRES-GFP.…”

“…Although BMP inhibition is absolutely required for neural induction to take place (De Robertis and Kuroda, 2004, Harland, 2000, Hemmati-Brivanlou and Melton, 1997, Linker and Stern, 2004), it is increasingly clear that other signals are also required (Stern, 2005). Known additional factors include FGFs, IGFs and Wnt inhibition, but even a combination of all of these factors is insufficient to mimic the effect of a node graft to the area opaca in the chick (de Almeida et al, 2008, Linker and Stern, 2004), suggesting that other factors are also involved.…”

Zeb2: A multifunctional regulator of nervous system development

“…It would be worthwhile to determine whether Zeb2 also recruits Smad7 to inhibit BMPSmad signalling during neural induction. Indeed, Smad7 has been shown to promote neural induction in the Xenopus and chick embryos (Bhushan et al, 1998;Casellas and Brivanlou, 1998;Chang and Harland, 2007;de Almeida et al, 2008). Weng et al (2012) demonstrated that Zeb2 not only acts as a transcriptional activator and repressor in the same cell, but that the transcriptional modulation of various differentiation regulators by Zeb2 is stage-dependent.…”

Regulation of programmed cell death during neural induction in the chick embryo

“…Several lines of evidence implicate BMP inhibition as an essential step in neural induction (de Almeida et al, 2008, Harland, 2000, Hemmati-Brivanlou and Melton, 1997, Linker et al, 2009, Linker and Stern, 2004, Marchal et al, 2009, Reversade et al, 2005, Stern, 2006, Vonica and Brivanlou, 2006. However chick epiblast cells cannot respond to BMP inhibitors unless they have previously been exposed for at least 5 hours to other signals from the organizer Stern, 2004, Streit et al, 1998).…”