Localization of myocyte enhancer factor 2 in the rodent forebrain: regionally-specific cytoplasmic expression of MEF2A

Abstract

The transcription factor myocyte enhancer factor 2 (MEF2) is expressed throughout the central nervous system, where four MEF2 isoforms play important roles in neuronal survival and differentiation and in synapse formation and maintenance. It is therefore somewhat surprising that there is a lack of detailed information on the localization of MEF2 isoforms in the mammalian brain. We have analyzed the regional, cellular, and subcellular expression of MEF2A and MEF2D in the rodent brain. These two MEF2 isoforms were co-expressed in virtually all neurons in the cortex and the striatum, but were not detected in astrocytes. MEF2A and MEF2D were localized to the nuclei of neurons in many forebrain areas, consistent with their roles as transcriptional regulators. However, in several subcortical sites we observed extensive cytoplasmic expression of MEF2A but not MEF2D. MEF2A was particularly enriched in processes of neurons in the lateral septum and bed nucleus of the stria terminalis, as well as in several other limbic sites, including the central amygdala and paraventricular nuclei of the hypothalamus and thalamus. Ultrastructural examination similarly revealed MEF2A-ir in axons and dendrites as well as MEF2A-ir nuclei in the lateral septum and bed nucleus of the stria terminalis neurons. This study demonstrates for the first time extensive cytoplasmic localization of a MEF2 transcription factor in the mammalian brain in vivo. The extranuclear localization of MEF2A suggests novel roles for MEF2A in specific neuronal populations.

Figure 1

Immunoblot analysis of MEF2A and MEF2D in the rat forebrain. Incubation with an antibody against MEF2A revealed a protein doublet of ∼ 55 kD in all tissues examined (A), while MEF2D migrated as a single band at around 55 kD (B); GAPDH was included as a loading control. Molecular weight standards are indicated at the right. (hip, hippocampus; sep, septum; str, striatum; ctx, cortex).

Figure 2

Immunhistochemical analysis of MEF2A and MEF2D in the rat forebrain. MEF2A (A-C) and MEF2D (D-F) expression were analyzed in the cortex (A,D), striatum (B,E), and the septum (C,F) of the adult rat by immunoperoxidase staining. In the cortex MEF2A- (A) and MEF2D-ir (D) were seen in nuclei of neurons across all cortical lamina. MEF2A- (B) and MEF2D-ir (E) nuclei were also seen throughout the striatum. In contrast, although MEF2D-ir is localized to nuclei in the septum (F), extensive MEF2A-ir processes are present in the LS/BNST (C). (*, lateral ventricle, cortical layers are indicated by Roman numerals). Scale bar = 500 μm.

Figure 3

MEF2A is expressed in neurons but not astrocytes. Cell type specific expression of MEF2A was assessed in the cortex (A-C) and striatum (D-E). MEF2A-ir (red) was almost always localized to nuclei in the cortex and striatum, although rare MEF2A-ir fiber-like structures can be seen in the medial (periventricular) striatum (E, arrows). Double-labeling experiments with antibodies against MEF2A (red) and the neuronal marker NeuN (green) reveals that virtually all NeuN positive cells express MEF2A in the cortex (A) and the striatum (B). In contrast, MEF2A-ir (red) does not colocalize with astrocytes labeled with GFAP (green) in the cortex (B) or the striatum (E). Counter-staining cortical (C) or striatal (F) sections with the nuclear stain DAPI (blue) reveals that virtually all large nuclei characteristic of neurons are MEF2A-positive (red), whereas in the smaller nuclei, typically associated with glial cells, MEF2A-ir is absent (C,F arrow head). Scale bar = 25 μm in A also applies to B, D, E; scale bar = 25 μm in C also applies to F.

Figure 4

MEF2A and MEF2D are colocalized in cortical and striatal neurons. Cortical (A-C) and striatal (D-E) sections were incubated with MEF2A (red) and MEF2D (green) antibodies. The merged images (C, F) demonstrate that MEF2A and MEF2D are co-expressed in virtually all cortical and striatal neurons, although the relative abundance of the two isoforms may vary somewhat, as reflected by the different shades of yellow. Scale bar = 25 μm.

Figure 5

Analysis of MEF2A expression in cortical interneurons. Cortical sections were stained for MEF2A (red) and one of three calcium binding proteins (CaBP) (green) that mark non-overlapping populations of cortical interneurons: parvalbumin (PV) (A-C), calbindin (CB) (D-F) or calretinin (CR) (G-I). Many, but not all (arrow heads) interneurons express MEF2A. Scale bar = 25 μm.

Figure 6

Extranuclear MEF2A-ir in neurons of the LS/BNST. Abundant MEF2A-ir fiber-like structures can be clearly seen in the LS/BNST of adult rats (A) and mice (B). Higher magnification images reveal a punctate perisomatic and neuritic MEF2A staining pattern (C). MEF2D (green) is exclusively seen in nuclei (C). Preincubation of the MEF2A antibody with the peptide that served as the immunogen obliterated the signal (D). Scale bar = 50 μm in A,B and D; scale bar = 25 μm in C.

Figure 7

Subcellular localization of MEF2A-ir in the LS/BNST. Immunoelectron microscopic analysis revealed MEF2A-ir in both axons and dendrites. Labeled axon terminals were particularly common (A, arrow). Preterminal myelinated (B, arrow) and unmyelinated (C, arrow) axons also contained MEF2A-ir. In addition, MEF2A-ir dendritic spines (D, arrow) and shafts (E, arrow) can be observed. Scale bar = 500 nm.

Figure 8

MEF2A-ir in axon terminals in the LS/BNST. Intensely-labeled axon terminals (arrows) can be seen in apposition to large, proximal dendrites (A) or cell bodies (B,C). Symmetric synapses on the MEF2A-ir terminals are indicated by arrowheads. (Scale bar in A = 1 μm; scale bar in B and C = 500 nm).