Too further validate the BRE-eGFP reporter animals and assess its sensitivity in comparison to staining tissues for nuclear pSmad1/5/8 by immuno-histochemistry, we stained adjacent tissue sections of P1 lungs, the developmental stage demonstrating maximal BRE-eGFP reporter activity, with anti-eGFP and anti-pSmad1/5/8 antibodies. As shown in Figure S4 , nuclear pSmad1/5/8 was detected in regions with intense eGFP immuno-staining demonstrating consistency between BRE-eGFP reporter activity and pSmad1/5/8 immuno-staining ( figure S4 ). Unfortunately, in our hands the anti-pSmad1,5,8 reagents we have analyzed so far were unable to detect pSmad1/5/8 in lung tissues from embryos before stage E19.5 and thus we were not able to confirm correlation of eGFP and pSmad1/5/8 at earlier developmental stages. A number of studies have indicated that the value of measuring pSmad1/5/8 as the sole indicator for canonical BMP pathway activation is questionable since presence of pSmad1/5/8 does not necessarily lead to transcriptional activity of BMP target genes. Canonical BMP pathway activity is known to be regulated by cell type-specific Smad-interacting transcription factors, co-activators and co-repressors. For example Tbx1 can bind to Smad1, interfere with Smad1/Smad4 interaction and decrease BMP transcriptional activity without affecting the amount of pSmad1/5/8 in the responding cell [42] . Furthermore, Leeuwis et al. [41] demonstrated that in the kidney of BRE-eGFP animals, whereas the expression pattern of BRE-eGFP exhibited excellent correlation with the expression of the BMP7 and the BMP target genes Id1, and Smad6, in contrast pSmad1/5/8 exhibited a brought expression pattern that did not correlate with the expression of BRE-eGFP, BMP7, Id1 or Smad6. Therefore, a safer assessment of canonical BMP pathway should combine analysis of several known BMP target genes and pSmad1/5/8 immuno-stainings when this is technically possible. Collectively, the analysis by Monteiro et al. [33] , Leeuwis et al. [41] and the results described herein indicate that the BRE-eGFP transgenic line reports quite faithfully activation of the canonical BMP pathway in-vivo and ex-vivo and thus it provides an additional method for assessing potential canonical BMP pathway activation.