Characterisation of a dual-colour retinoic acid reporter system for analysing cells and embryos
Vitamin A is an important component in the diet as its metabolites, the retinoids, play key roles in a vast range of cellular functions, from production of germ cells, to embryonic development and right through until adulthood. The function of retinoids, in particular retinoic acid (RA), is especially important during early embryonic development, where it is responsible for many different key developmental events. Some of the processes controlled by RA include brain region patterning, Hox gene expression, axis establishment and somite formation. Here, we aimed to characterise the expression pattern of retinoic acid in the early murine embryo and isolate cell populations from a range of RA concentrations to analyse the mRNA expression. To do this, we used a transgenic mouse line which expressed a reporter plasmid that was able to show, through the expression of two fluorescent proteins, areas of high RA concentration and area of low RA concentration. We tested the function of this reporter system in vitro, using cell lines which were transfected with the plasmid and exposed to RA in their growth media. This worked showed a somewhat does-dependent response from the reporter system expressing the fluorescent proteins. We then imaged transgenic embryos at various stages of early development, to ascertain the areas of RA expression and repression. Here, we saw fluorescent protein expression patterns that indicated both high and low concetrations of RA. Using this information, we dissociated transgenic E8.5 embryos and sorted the cells based on their levels of expression of the two fluorescent proteins, as well as by tissue type, which had been marked with antibodies. mRNA was extracted from these populations and PCR was performed to identify the presence of Hox genes and to see any difference in expression patterns across the various cell populations.