Gene-expression analysis was carried out at a few key time points in the buds, which receive the ‘AB signal’, and in leaves and stems, which might play a role in generating and transporting the signal. Our resequencing study reported here and those reported elsewhere Cinoxacin indicate that non-synonymous mutations of CITED2 can be observed in patients with congenital heart disease, and that these mutations tend to cluster in the SRJ domain. Cellbased assays investigating some of these variants have indicated that they can affect HIF1A-repression and/or TFAP2-coactivation functions of CITED2. The SRJ domain, although highly conserved in placental mammals, is substantially abbreviated in marsupials and in monotremes, and is absent in other vertebrates. Thus, the region may have appeared relatively recently in evolutionary terms and may conceivably be of relevance to differences in cardiac development and structure between placental mammals and other vertebrates. Structurally, the SRJ region is predicted to be disordered and potentially functions as a flexible linker. The T166 residue within this domain is predicted to be a target of proline directed kinases, and our studies indicated that the T166 residue can be phosphorylated by MAPK1, and that activation of MAPK1 promoted co-activation function. Moreover cell-based studies indicated that the T166N mutation had a deleterious effect on TFAP2 co-activation function, and on the ability of CITED2 to promote ES cell proliferation in the absence of LIF. Surprisingly, our in vivo study of mice carrying the T166N 3,4,5-Trimethoxyphenylacetic acid variant and the deletion of the SRJ domain indicate that in mice, under normal laboratory conditions, neither the complete deletion of the SRJ domain nor the introduction of a variant which could potentially lead to the loss of a phosphorylation site of CITED2 are detrimental to its function. In a single Cited2 2/MRG1 embryo out of 75 that we studied, we observed an ectopia cordis phenotype. Since ectopia cordis is not a phenotype which has previously been associated with the loss of Cited2, and this embryo has normal adrenal glands, it is most unlikely that it is a consequence of the loss of the SRJ domain. Animals of this genotype are also viable and fertile. However, in vitro data indicate that the T166N mutation can have functional significance, and previous studies have indicated that other SRJ mutations also affect its function. It is possible that the partial impairment of CITED2 function revealed by in vitro experiments is insufficient to affect development. Taken together, the results obtained from mouse studies indicate that the SRJ domain is dispensable during mouse cardiac development and for viability and fertility. On the other hand, three independent human studies show that non-synonymous mutations, predominantly clustering in the SRJ domain, are mainly observed in patients with CHD and not in controls suggesting that this region is important for normal cardiac development. How can we explain these divergent observations? One possibility is that the SRJ is indeed dispensable for mammalian heart development, and that the observations from patients may be misleading. Supporting this idea, there is considerable lack of conservation in this domain between placental and non-placental mammals. Furthermore, the mutations in the SRJ do not significantly affect the disordered nature of the domain, indicating that it may be able to accommodate mutations without adversely affecting the overall structure and function of the protein. Moreover, in no case has it been shown that the mutation has either arisen de novo or been transmitted from an affected parent. Another possibility that partially reconciles the mouse and human observations is that variants found clustered in the SRJ in CHD patients may not, by themselves.