Jennifer Mansfield
Harvard Medical School
Department of Genetics
77 Avenue Louis Pasteur
Boston, MA
02115

(617) 432-6532
jmansfie@genetics.med.harvard.edu

microRNAs and vertebrate development

microRNAs (miRNAs) are a class of short (~22nt) non-coding RNA molecules that down-regulate expression of their mRNA targets. Since their discovery as regulators of developmental timing in C. elegans, hundreds of miRNAs have been identified in both animals and plants; however, only a small number have known functions. As a first step toward understanding their functions, it is important to understand where and when they are expressed. We developed a transgenic assay for visualizing detailed spatial and temporal expression patterns of miRNAs within the mouse embryo, with the goal of identifying miRNAs with potential developmental roles.

We generated a series of miRNA-responsive reporter constructs designed to detect the presence of specific miRNAs (Figure 1). Each transgene contains a constitutively expressed reporter (lacZ) bearing sequences complementary to a given miRNA within the 3’UTR. In cells lacking the specific miRNA that the transgene is designed to detect, the transgene RNA should be stable and hence ß-galactosidase (ß-gal) should be expressed (Figure 1a). In contrast, in cells that express the miRNA, its perfect complementarity to sequences in the 3’UTR of the transgene is expected to target the transgene mRNA to the RNAi pathway, resulting in the absence of ß–gal activity (Figure 1b). Thus in transgenic embryos, tissues stained for ß–gal will appear white when the target miRNA is present and will be blue everywhere else.

Using this system, we have identified several miRNAs with restricted expression patterns in mouse development. For example, we found that miR-1 is specifically expressed within the embryonic heart. As shown in Figure 2a, in an embryo bearing a transgene containing miR-1 complementary sites, lacZ is downregulated specifically within the heart. Moreover, the chambers of the heart lack ß-gal activity, while the outflow tract (arrow) remains blue, revealing a restricted expression pattern of miR-1 within a subset of heart structures. In contrast, in an embryo bearing a transgene that lacks known miRNA complementary sites, lacZ is ubiquitously expressed, as expected (Figure 2b).