Our ongoing analysis of Nodal signaling involves the use of conditional mutagenesis: we have generated a conditional """"""""floxed"""""""" allele, which is being used in conjunction with transgenic lines expressing Cre recombinase in various lineages of the early embryo. We have found that primitive endoderm specific Nodal gene deletion starting at the earliest stages of post-implantation development leads to failure of subsequent anterior-posterior axis formation due to defects in distal visceral endoderm migration. While this is an important finding, we could not determine whether the observed defect is cell autonomous to the visceral endoderm or arises due to loss of visceral endoderm expression combined with Nodal heterozygosity in the remainder of the embryo. The conventional approach to Cre/loxP conditional mutagenesis involves establishing Cre mice with a null allele for the gene under study, which are then bred to mice carrying the floxed allele of that gene. Thus the resulting informative offspring are heterozygous in all tissues and homozygous mutant in the tissues expressing Cre. We attempted to establish Cre strains that carried the floxed allele, rather than the null allele. While this is theoretically possible for Cre strains not expressed in the germ line, in practice it can be difficult. In fact all Cre mice we derived were mosaic in their germ line and ultimately not useful. Therefore, we are taking a different approach to address the question of cell autonomy. We made the unexpected discovery that some Nodal heterozygotes display the same distal visceral endoderm migration defect. In their case, though, this must represent a delay in the process that they are able to recover from, as most heterozygotes do develop normally. This finding provides evidence that the defect we are seeing in the conditional mutant results from an overall reduction in Nodal expression, rather than being due to loss of a cell autonomous function within the visceral endoderm. To follow up this finding we are generating embryos that have half as much (or less) Nodal in specific parts of the embryo, and not in others. This should allow us to determine the overall threshold level required for normal visceral endoderm migration. This work has important implications for understanding where and when Nodal signals are essential for establishing the earliest patterning events in mouse development. Additional work on Nodal in our lab is addressing the epigenetic regulation of its expression. Histone modifications have been proposed to function as an epigenetic code independent from their role in ongoing chromatin processes, with trimethylated lysine 4 on histone H3 (H3K4me3) mainly found in active promoter regions and trimethylated lysine 27 (H3K27me3) marking genes for repression by polycomb proteins. The presence of both these modifications in promoters of developmentally important genes in embryonic stem (ES) cells recently has been suggested to function as an epigenetic signal marking these genes for transcriptional activation later in development. Nodal expression is regulated by an intronic enhancer, which is activated by the Nodal signaling pathway itself, and upstream distal enhancers. These regulatory elements have well defined roles in regulating Nodal expression at specific developmental stages and in specific cell lineages. Thus, the Nodal locus provides an important test case for studying epigenetic control of developmental gene expression. Given the central role of H3K4me3 and H3K27me3 in epigenetic mechanisms, we looked for these modifications within the Nodal enhancer domains both in ES cells, which actively transcribe Nodal, and in Nodal negative extraembryonic endoderm (XEN) cells. H3K4me3 was detected in the intronic enhancer and promoter region in ES cells but was absent from these regions in XEN cells, in agreement with this modification marking active promoters. In the upstream enhancer domains, we detected H3K4me3 in both ES and XEN cells, but the H3K27me3 modification was found only in XEN cells. These findings suggest that the specific combination of modifications found in the upstream domains acts as an epigenetic signal dictating gene expression status. Our work has important implications for how Nodal expression may be regulated during development.
| Lowe, L A; Yamada, S; Kuehn, M R (2001) Genetic dissection of nodal function in patterning the mouse embryo. Development 128:1831-43 |
| Fujinaga, M; Lowe, L A; Kuehn, M R (2000) alpha(1)-Adrenergic stimulation perturbs the left-right asymmetric expression pattern of nodal during rat embryogenesis. Teratology 62:317-24 |
| Lowe, L A; Yamada, S; Kuehn, M R (2000) HoxB6-Cre transgenic mice express Cre recombinase in extra-embryonic mesoderm, in lateral plate and limb mesoderm and at the midbrain/hindbrain junction. Genesis 26:118-20 |
| Pfendler, K C; Yoon, J; Taborn, G U et al. (2000) Nodal and bone morphogenetic protein 5 interact in murine mesoderm formation and implantation. Genesis 28:14-Jan |
| Campione, M; Steinbeisser, H; Schweickert, A et al. (1999) The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping. Development 126:1225-34 |
