Visualization of effects of estrogen to the brain by using GFP

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Estrogen plays important roles in regulating structure and function of many neuronal systems, but detailed mechanisms of the regulation are still not clear. We have studied about these unknown mechanisms by two way using green fluorescent protein (GFP) and its spectral variants, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) that are useful for imaging in living cells or tissues. To investigate the relationships between the loci expressing functions of ERα and that of ERβ, we analyzed the subnuclear distribution of ERα and ERβ in response to the ligand in single living cells using fusion proteins labeled with fluorescent proteins. In a hypothalamic cell line, RCF12 cells, ERα and ERβ were co-localized in the nucleus both before and after the addition of 17β-estradiol. Upon addition of the ligand, distribution of ERs was changed to discrete pattern rapidly, suggesting that ligand binding causes redistribution of the receptors to nuclear sites to be activated. Deletion analysis showed that this ligand-dependent redistribution of ERs might be occurred through complex interactions via broad part of the receptor including at least the latter part of activation function-1, DNA binding domain, nuclear matrix binding domain and activation-2/ligand binding domain. In addition, to know which nuclear components were associated with the discrete cluster of ERs formed in response to the hormone treatment, immunofluorescence study against several nuclear proteins was examined. ERs were mostly colocalized with hyperacetylated histone H4 and a chromatin remodeling protein, Brg-1 , indicating that most of the ERs clusters might be involved in structural change of chromatin. Furthermore, mobility of ERα within the nucleus was analyzed by fluorescence recovery after photobleaching (FRAP) technique. Interestingly, the discrete cluster of ERα was rapidly and dynamically exchanged at second order suggesting that the mobility of ligand activated ERα was required to the transcriptional regulation of the receptor. On the other hand, to visualize ERα expressing neurons, we generated transgenic mice expressing GFP under the control of the ERα promoter. In 3 independent transgenic lines, GFP positive neurons were observed at the areas previously reported to express ERα mRNA in rat, which included lateral septum, bed nucleus of stria terminalis, amygdala, hypothalamus regions, and in these areas, GFP signals were mostly overlapped with ERα immnoreactivity. GFP fluorescence was observed in axons and dendrites as well as in cell bodies of the neurons. Thus, the ERα promoter-GFP transgenic mice will be useful to analyze development and plastic change of network and structure of ERα expressing neurons in living brain tissue and possibly to identify specific genes of ERα expressing neurons by selective collection of GFP-positive neurons.
日本組織細胞化学会の論文

Visualization of effects of estrogen to the brain by using GFP

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