Expression of macrophage migration inhibitory factor in the mouse neocortex and posterior piriform cortices during postnatal development

Abstract

Macrophage migration inhibitory factor (MIF) functions as a pleiotropic protein, participating in a vast array of cellular and biological processes. Abnormal expression of MIF has been implicated in many neurological diseases, including Parkinson's disease, epilepsy, Alzheimer's Disease, stroke, and neuropathic pain. However, the expression patterns of mif transcript and MIF protein from the early postnatal period through adulthood in the mouse brain are still poorly understood. We therefore investigated the temporal and spatial expression of MIF in the mouse neocortex during postnatal development in detail and partially in posterior piriform cortices (pPC). As determined by quantitative real-time PCR (qPCR), mif transcript gradually increased during development, with the highest level noted at postnatal day 30 (P30) followed by a sharp decline at P75. In contrast, Western blotting results showed that MIF increased constantly from P7 to P75. The highest level of MIF was at P75, while the lowest level of MIF was at P7. Immunofluorescence histochemistry revealed that MIF-immunoreactive (ir) cells were within the entire depth of the developed neocortex, and MIF was heterogeneously distributed among cortical cells, especially at P7, P14, P30, and P75; MIF was abundant in the pyramidal layer within pPC. Double immunostaining showed that all the mature neurons were MIF-ir and all the intensely stained MIF-ir cells were parvalbumin positive (Pv +) at adult. Moreover, it was demonstrated that MIF protein localized in the perikaryon, processes, presynaptic structures, and the nucleus in neurons. Taken together, the developmentally regulated expression and the subcellular localization of MIF should form a platform for an analysis of MIF neurodevelopmental biology and MIF-related nerve diseases.

Fig. 1

Expression profile of MIF during postnatal development. a Relative qPCR for mif mRNA in the mouse neocortex during postnatal development. The qPCR measurements from three animals were averaged to compare the mRNA level for each age. Mif transcript was increased progressively from P0 to P30 and then declined dramatically during development. b Using Western blotting, the MIF antibody recognized a band of 12 kDa specially. The band for β-Tubulin was used as a control for normalizing MIF expression at different developmental ages. Three samples were collected for each timepoint, and Western blotting was performed three times independently. c Densitometry measurements from three independent experiments were averaged for each developmental age and normalized to P75. During postnatal development, MIF protein was detected at birth and expressed at the lowest level at P7. Data were presented in bar graphs showing mean values ± SEM, ns non significant, **p < 0.01

Fig. 2

MIF expression in adult nervous tissues. a A single band was seen with protein extracts from the adult mice neocortex, hippocampus, spinal cord, and ischiadic nerve with β-Tubulin as the endogenous control. Signal of MIF was hardly detected in ischiadic nerves. Three samples were collected from different litters for each part. b Densitometric analysis of MIF in the adult neocortex, hippocampus, spinal cord, and ischiadic nerve normalized to the neocortex. Compared with MIF protein in the neocortex, MIF protein of the hippocampus increased 0.3-fold and MIF protein of ischiadic nerves decreased 98 %. Data were presented in bar graphs showing mean values ± SEM, ns non significant, *p < 0.05, ***p < 0.001 compared to the neocortex. NC Neocortex; HP Hippocampus; SP Spinal cord; IN Ischiadic nerve

Fig. 3

Immunofluorescence histochemistry for MIF in the neocortex at P0, P3, P7, P14, P30, and P75 developmental timepoints. a–i MIF was detected with polyclonal rabbit anti-MIF antibody (green). Cortical layers were numbered in roman digits. IZ intermediate zone; VZ/SVZ ventricular zone/subventricular zone; WM white matter. c–f Two images were photomerged in a montage to form one image by Adobe Photoshop CS4. g–i Nuclei were counterstained with PI (red). Enlargement of underlayer VI at P14 (g), P30 (h) and P75 (i). Scale bar 100 μm (Color figure online)

Fig. 4

Immunofluorescence localization of MIF in the neocortex of layer I and II/III at P0 (a) and layer I/II (b), III/IV (c), V/VI (d), VI/WM (e) at P75. Coronal sections of the brains were stained with MIF antibody (green) and PI (red). Cortical layers were numbered in roman digits. WM white matter. Scale bar 20 μm (a); 50 μm (b–e) (Color figure online)

Fig. 5

Subcellular localization of the MIF protein in transfected neurons. a Schematic diagram of the CAG::MIF–MYC fusion construct, CAG::EGFP–MYC fusion construct and the CAG::EGFP construct. b Transient expression of CAG::EGFP construct in transfected neurons. Embryonic brains were electroporated with CAG::EGFP expressing vector at E15.5, followed by fixation at P7. Coronal sections of the brains were stained with GFP antibody (green) and PI (red). c Transient expression of CAG::EGFP–MYC fusion construct in transfected neurons. Embryonic brains were electroporated with CAG::EGFP–MYC expressing vector at E15.5, followed by fixation at P7. Coronal sections were stained with GFP antibody (green) and PI (red). d Transient expression of CAG::MIF–MYC fusion construct in transfected neurons. Embryonic brains were electroporated with CAG::MIF–MYC expressing vector plus CAG::EGFP expressing vector at E15.5, followed by fixation at P7. Sections were incubated in mouse anti-c-MYC and then exposed to Alexa Fluor 568 donkey anti-mouse IgG. Nuclei were counterstained with DAPI. MIF-MYC was represented by false color green (red channel), and nuclei were represented by false color red (blue channel). Scale bar 50 μm (Color figure online)

Fig. 6

Double-label fluorescent immunohistochemistry for simultaneous detection of MIF and presynaptic marker synaptophysin (a) or postsynaptic marker PSD95 (b) in layer I of the neocortex at P75. Coronal sections of the brains were stained with MIF antibody (green) along with various antibodies against synaptophysin (red) and PSD95 (red). Synap Synaptophysin. Scale bar 2 μm (Color figure online)

Fig. 7

MIF staining in posterior piriform cortex at P14, P30, and P75. a–c MIF-ir cells (green) were predominately located in pyramidal layers. Nuclei were counterstained with PI. d Enlargement of the above smaller boxed area—pyramidal layer. e Enlargement of the above larger boxed area—polymorph layer. The asterisks indicated the pia surface of the cortex. ml molecular layer; pyl pyramidal layer; pol polymorph layer. Scale bar 100 μm (a–c); 20 μm (d); 50 μm (e) (Color figure online)

Fig. 8

Double-label fluorescent immunohistochemistry for simultaneous detection of mature neurons marker NeuN (red) and MIF (green) in the neocortex at P75. a NeuN positive cells throughout the entire depth of the neocortex were MIF-ir and displayed varied MIF staining. b Enlargement of the neocortex within layers II/III. Arrows indicated intensely stained MIF and NeuN positive cells. The arrowhead indicated a intensely stained MIF and slightly stained NeuN positive cell. Scale bar 300 μm (a); 50 μm (b) (Color figure online)

Fig. 9

Double-label fluorescent immunohistochemistry for simultaneous detection of parvalbumin (red) and MIF (green) in the neocortex at P75. a All the intensely stained MIF-ir cells were Pv +. b Enlargement of the neocortex within the layer V. c Total number of co-stained cells (red, co-loc) in intense MIF-ir or intense Pv + cells throughout layers I–VI of all eight analyzed hemispheres. d Proportion of co-stained cells. The amount of double-labeling cells were depicted as a percentage of total intense MIF-ir or intense Pv + cells throughout layers I–VI. Data were presented in bar graphs showing mean values ± SEM, n = 8. Pv parvalbumin. Scale bar 100 μm (a); 10 μm (b) (Color figure online)