We present a system by which diverse transcriptional reporter elements, downstream of different signal transduction pathways, elicit cellular fluorescence in an agonist-dependent and agonist-selective fashion in live cells and embryos. By employing the constitutively active Rosa26 gene and analyzing its upstream promoter sequences, we made successive deletions with the intention of preserving the inherent ‘openness’ of the locus and its constitutive activity, while diminishing core promoter function sufficiently to provide a low background of transcription in the absence of signal pathway activity. The −228 deletion endpoint satisfied these criteria, whereas the −60 deletion retained high constitutive activity. Fortunately, we discovered that insertion of all signaling elements studied here at −60 resulted in a marked diminution of basal activity. Not unexpectedly, the different −228 and −60 constructs provided slightly different results in terms of basal and induced activity with different regulatory element insertions. We suggest that further use of this model with new signaling sentinels would be best served by trying both the −60 and −228 deletions in ES cells. The RMCE method makes it relatively simple to swap a desired signaling sentinel into the Rosa26 locus and the exchanged clones can be screened readily for proper insertion and signal responsiveness.
As noted in the Introduction and Results, signaling reporters have been generated for many of the pathways studied here. However, to date they have utilized conventional transgene technology, where transgenes are inserted at random locations throughout the genome. The random locations result in position effects, such that different transgenic lines with the same construct do not yield identical signaling readouts and might also exhibit variegated expression. Moreover, many of the existing reporters have employed lacZ or luciferase and thus are used on fixed or lysed cells. By utilizing fluorescent reporters integrated into the same locus, for different signaling sentinels, we eliminate position effects and allow systematic modification of response elements in a constant genomic location. By demonstrating that the system works with Cerulean, mCherry and mOrange reporters, it paves the way for crossing distinct signaling reporter lines of mice to create heteroallelic animals. This would allow different signaling pathways to be assessed simultaneously in live animals. We chose fluorescent reporters with their native half-life determinants, rather than high-turnover derivatives, because we felt that the latter would be less sensitive overall and we wished for maximum signal to allow visualization of the earliest times of induction and in intact embryos (e.g. ; ; ).
Along these lines, we were able to successfully create sentinels that distinguish BMP and activin A signaling, despite the fact that their pathways possess components in common. Whereas the BMP sentinels exhibited uniform fluorescence shift profiles for the induced population, the activin A sentinel exhibited a much broader profile in the induced population. Further analysis of the AR8 element will be required to determine its unusual profile of activity. However, because the function of the AR8 element was assessed here in an allelic fashion with diverse other constructs that gave more uniform cellular response shifts, we can attribute the difference to unexpected factors in the composition of the AR8 element, i.e. additional factors that bind. Our approach allows for subsequent dissection of regulatory elements and optimization, in a systematic fashion, by performing RMCE with variant AR8 elements at the originally targeted locus.
Interestingly, previous studies of transfected or injected reporter constructs found that Mix.2
-based AR elements required co-transfected or injected Forkhead activin signal transducer (Fast-1) in order to exhibit TGFβ- or activin-based induction (Weisberg et al., 1998
; Yeo et al., 1999
; Osada et al., 2000
). By contrast, our AR8 reporter did not need additional Fast-1 for highly specific activin A responsiveness. We attribute the difference to the monoallelic nature of our reporter and its apparent ability to function sufficiently with endogenous Fast-1 (Sirard et al., 2000
) or with Fast-2 (Nagarajan et al., 1999
), in comparison with transfected or injected constructs that involve many DNA copies per cell. This emphasizes the effectiveness of our targeted integration approach, in contrast to transfected or transgenic reporters that typically involve multiple gene copies per cell. Additionally, the ability of SB431542 to inhibit the activin response shows that the response is dependent on receptor activation.
We note that by having the same signaling reporter in ES cells and mice, it is now possible to directly compare inductive signaling events seen in particular lineages and stages in development with signaling events occurring during in vitro stem cell differentiation. Similarly, signaling that occurs during homeostasis and regenerative responses in vivo should be informative for the maintenance and further manipulation of cell types generated in vitro.
We based our Rosa26 promoter deletion endpoints on landmarks that are conserved between mouse and human. Thus, it should be straightforward to apply the same promoter deletion-substitution approach to the Rosa26 locus in human ES cells and thereby provide a sensitive means by which cell programming can be monitored and guided in living human ES cells. The high signaling sensitivity of most of the sentinels we created and their high signaling specificity suggests that the −60 and −228 deletion sites for the Rosa26 locus should be useful for the assessment of diverse types of signal response elements and pathways in vivo and in vitro.