Dev Neurobiol. 2015 Mar;75(3):232-48.

Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B-GFP transgenic mice.

 

Mei-Shu Chen1 and Ing-Ming Chiu1,2,3*

1Division of Regenerative Medicine, Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli 35053, Taiwan, R.O.C.,

2Department of Life Sciences, National Chung-Hsing University, Taichung 40227, Taiwan, R.O.C.,

3Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA

*Corresponding author: Ing-Ming Chiu

Address: Division of Regenerative Medicine, Institute of Cellular and System Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan.

Phone: 886-37-246166 ext. 37501

Fax: 886-37-587408

E-mail: ingming@nhri.org.tw

 

Abstract

FGF1 is involved in multiple biological functions and exhibits the importance in neuroprotective effects. Our previous studies indicated that, in human brain and retina, the FGF1B promoter controlled the expression of FGF1. However, the exact function and regulation of FGF1 in brain is still unclear. Here, we generated F1B-GFP transgenic mice that expressed the GFP reporter gene under the control of human FGF1B promoter (from nucleotides -540 to +31). Using the fresh brain sections of F1B-GFP transgenic mice, we found that the F1B-GFP cells expressed strong fluorescent signals in the ventricular system throughout the brain. The results of immunohistochemistry further showed that two distinct populations of F1B-GFP+ cells existed in the brains of F1B-GFP transgenic mice. We demonstrated that one population of F1B-GFP+ cells was ependymal cells, which distributed along the entire ventricles, and the second population of F1B-GFP+ cells was neuronal cells that projected their long processes into multiple directions in specific areas of the brain. The double labeling of F1B-GFP+ cells and tyrosine hydroxylase indicated that a subpopulation of F1B-GFP+-neuronal cells was dopaminergic neurons. Importantly, these F1B-GFP+/TH+ cells were distributed in the main dopaminergic neuronal groups including hypothalamus, ventral tegmental area and raphe nuclei. These results suggested that human FGF1B promoter was active in ependymal cells, neurons and a portion of dopaminergic neurons. Thus, the F1B-GFP transgenic mice provide an animal model not only for studying FGF1 gene expression in vivo but also for understanding the role of FGF1 in neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease.

Keywords: FGF1; human FGF1 promoter; transgenic mice; dopaminergic neurons; adult brain

PMID: 25104610

 

 

Introduction

FGF1 plays an important role in neuroprotective activities of neuron injury or neurological disorder (Date et al., 1990; Cuevas et al., 1994; Kimura et al., 1994; Teng et al., 1998). The human FGF1 gene is under control by at least four promoters, named 1A, 1B, 1C and 1D (Wang et al., 1991; Myers et al., 1995a; Hsu et al., 2009). Our previous results showed that the 571-bp (nucleotides -540 to +31) sequence upstream of human FGF1B transcription initiation site is sufficient to drive the expression of a heterologous luciferase reporter in cultured cells (Myers et al., 1995b). In addition, the human and mouse FGF1 showed highly evolutionary conservation in their protein structures and biological functions (Patrie et al., 1997). Therefore, in this study, we used the mouse as a study platform to elucidate the regulation of human FGF1B promoter in vivo.

 

Results

We generated a transgenic mouse line named F1B-GFP transgenic mouse that expressed GFP reporter under the control of human FGF1B promoter (nucleotides -540 to +31). Through the analyses of reverse transcription-polymerase chain reaction (RT-PCR), fluorescence detection in fresh brain sections and immunohistochemistry (IHC), we showed that human FGF1B promoter was successfully activated in the brains of F1B-GFP transgenic mice and dictated the expression of GFP reporter gene.

 

Figure 1Figure 1 Human FGF1B promoter was activated in transgenic mouse brain. The IHC results of brain section staining with the GFP antibody showed that F1B-GFP+ cells were detected in two different populations. The first population was in the ventricular system including LV (A), D3V (B), 3V (B and C), and Aq (D). The yellow arrows indicated that the second population was distributed in specific brain areas, including thalamus (B) and hypothalamus (C). Panels (A’)-(D’) were IHC results, double labeled with the GFP antibody and DAPI. Scale bar: 200 μm. Abbreviations: LV: lateral ventricle; D3V: dorsal third ventricle; 3V: third ventricle; Aq: aqueduct.

 

According to the localization and cell morphology, the F1B-GFP+ cells could be separated into two different populations in the brains of F1B-GFP transgenic mice [Fig. 1]. The first population of F1B-GFP+ cells was found along the ventricular system, such as lateral ventricle (LV) [Fig. 1 (A) and (A’)], dorsal third ventricle (D3V) [Fig. 1 (B) and (B’)], third ventricle (3V) [Fig. 1 (B) and (B’); (C) and (C’)] and aqueduct (Aq) [Fig. 1 (D) and (D’)]. The second population of F1B-GFP+ cells was located in specific brain areas, including thalamus [Fig. 1 (B) and (B’) and hypothalamus [Fig. 1 (C) and (C’)].

Two ependymal cell markers, S100β and Vimentin, labeled the brain sections of F1B-GFP transgenic mice. The IHC results confirmed that the first population of F1B-GFP+ cells was ependymal cells [Fig. 2]. We quantified the whole ventricular system and found that F1B-GFP+-ependymal cells were highly populated in the ventricles of forebrain, including LV, D3V and 3V [Fig. 2 (A) – (C)].

The double labeling of GFP and NeuN (neuron-specific marker) antibodies indicated that the second population of F1B-GFP+ cells was neurons, with long cell processes projecting into multiple directions [Fig. 3 (A) and (B)]. The F1B-GFP+-neruonal cells were widely distributed in transgenic mouse brain, including nucleus of the vertical limb of the diagonal band (VDB), hypothalamus, posterodorsal and posteroventral parts of medial amygdaloid nucleus (MePD and MePV), ventrolateral periaqueductal gray (VLPAG), ventral tegmental area (VTA), raphe nucleus and medulla oblongata.

The raphe nucleus has been implicated in Alzheimer’s disease (Michelsen et al., 2008). Studies also indicated that FGF1 is involved in Alzheimer’s disease (McLay et al., 2001; Mashayekhi et al., 2010; Lou et al., 2012). Administration of human FGF1 into animal model of Alzheimer’s disease significantly improved the learning and memory abilities, and attenuated neurodegeneration (Lou et al., 2012). Thus, our results showed that the human FGF1B promoter was activated in the neurons in raphe nucleus strongly corroborated the notion that FGF1 plays an important role in Alzheimer’s disease.

 

Figure 2Figure 2 The F1B-GFP+ cells were ependymal cells and distributed in the entire ventricular system of F1B-GFP transgenic mouse brain. The brains of F1B-GFP transgenic mice were stained with ependymal cell markers, S100β [(A) and (B)] and Vimentin [(C) and (D)] antibodies. Scale bar: 25 μm. Abbreviations: LV: lateral ventricle; 3V: third ventricle; D3V: dorsal third ventricle; CC: central canal.

 

In addition, we found that a subpopulation of F1B-GFP+-neuronal cells was dopaminergic neurons [Fig. 3 (C)]. These F1B-GFP+/tyrosine hydroxylase (TH)+ cells were distributed in hypothalamus, VTA, raphe nucleus and medulla oblongata [Fig. 3 (C)].

Previous studies indicated that FGF1+ cells in the medulla oblongata were cholinergic neurons in rat brain (Toyoda et al., 2006). However, double labeling of the GFP and choline acetyltransferase (ChAT) antibodies on brain sections from F1B-GFP transgenic mice showed that F1B-GFP+ cells were ChAT negative in medulla oblongata [Fig. 3 (D)]. The controversy that existed between the two different results regarding the FGF1 expression in cholinergic neurons remains to be further explored.

In summary, we provided the first evidence that human FGF1B promoter was activated in ependymal cells and dopaminergic neurons in the adult brains of the F1B-GFP transgenic mice. We also showed the specific regulation of human FGF1B promoter in mouse brain. Therefore, the F1B-GFP transgenic mouse line could provide an animal model to study the regulation of FGF in vivo and human neurological diseases such as Alzheimer’s disease and Parkinson’s disease.

 

Figure 3Figure 3 A second population of F1B-GFP+ cells was neurons; these neurons in medulla oblongata could be found in dopaminergic neurons but not in cholinergic neurons. (A) The medulla oblongata of F1B-GFP transgenic mice was double stained with GFP (green) antibody and the mature neuron marker NeuN (red). (B) The results of (A) merged with DAPI staining. Scale bar: 250 μm. (C) A subpopulation of F1B-GFP+/TH+ cells was found in medulla oblongata (arrows). (D) No F1B-GFP+/ChAT+ cells could be detected in brain stem. Scale bar: 25 μm. Abbreviations: CC: central canal.

 

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