QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hirai, S.-i. Articles by Ohno, S. Search for Related Content PubMed PubMed Citation Articles by Hirai, S.-i. Articles by Ohno, S. Social Bookmarking                         What's this?

MAPK-upstream protein kinase (MUK) regulates the radial migration of immature neurons in telencephalon of mouse embryo

Department of Molecular Biology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan

View larger version (105K): [in a new window]   Fig. 1. The detection of MUK protein in developing mouse telencephalon. (A) Western blot analysis of proteins extracted from 293T cells (–), 293T cells overexpressing MUK (M) and E16 telencephalic vesicles (T), using affinity-purified rabbit antibody against the C-terminal 276 amino acids (left panel) or normal rabbit IgG as a control (right panel). The arrowhead indicates the position of a 120 kDa protein (MUK). (B) Sagittal sections of paraffin wax-embedded E16 embryo head were immunostained (blue) using the anti-MUK antibody, alkaline phosphatase-conjugated secondary antibody and BM purple. Positions of the telencephalon (T), diencephalon (Dc), midbrain (Mb), cerebellar primordium (Cb), medulla oblongata (MO), spinal cord (SC), dorsal root ganglia (DRG) and lateral ventricle (LV) are indicated. The insert is a control staining with normal rabbit IgG. (C) Higher magnification of the telencephalic region in B, showing predominant expression of MUK in the subplate, intermediate zone and subventricular zone. (D-I) Paraffin wax-embedded sections were prepared of the heads of E11, E13, E14 and E15 embryos and the isolated brain of an E18 embryo or 5-day-old mouse (P5) and used for the immunostaining. Distinct layers observed in the cortical region, neuroepithelium (NE), ventricular zone (VZ), pre-plate (PP), subventricular zone (SVZ), intermediate zone (IZ), subplate (SP), cortical plate (CP), marginal zone (MZ) and neocortex consisting of six presumptive layers (I-VI) are indicated. Scale bars: 500 µm.

View larger version (97K): [in a new window]   Fig. 2. The activation of JNKs at the intermediate zone. (A) Western blot analysis of proteins (10 µg) extracted from NIH3T3 cells with UV radiation (UV), serum stimulation (S) or no treatment and E16 telencephalic vesicles (T), using antibodies against JNK (a), p38 (b), ERK (c) and the active/phosphorylated form of each enzyme, p-JNK, p-p38 and p-ERK, as indicated. Arrowheads indicate protein bands corresponding to each enzyme. (B) Immunohistochemical staining of frozen sections using alkaline phosphatase-conjugated secondary antibody. Serial sagittal sections were prepared with the heads of E16 embryos (a-e) and the isolated brain of an E18 embryo (f,g) and stained with the anti-MUK antibody (a,f), anti-active MAPKs, p-JNK (b,g), p-p38 (c), p-ERK (d) or control normal rabbit IgG (e). Arrows indicate the intermediate zones stained with antibodies against MUK, p-JNK or p-p38. The arrowhead indicates the cortical plate stained with anti-p-ERK antibody. Ventricular zone (VZ), intermediate zone (IZ), and cortical plate (CP) are indicated. (C) Frozen sections were prepared from E16 telencephalon (T), and the cortical region was dissected into three layers, mainly composed of the ventricular zone (V), intermediate zone (I), and cortical plate (C). Proteins (10 µg) in each layer were separated by SDS-PAGE and analyzed by western blotting using antibodies against MUK (a), p-JNK (b), JNK (c), MAP2 (e) or vimentin (f), or stained with Coomassie Brilliant Blue (d). (D) JNK activity was detected by in-gel kinase assay using Jun protein as a substrate. The protein samples are the same as in A,C. Arrowheads indicate the positions of p46 and p55 JNKs. The asterisk indicates constitutively active protein kinases also found in unstimulated NIH3T3 cells (Hirai et al., 1996).

View larger version (92K): [in a new window]   Fig. 3. Constitutive expression of MUK arrests cells at the subventricular/intermediate zone. (A) The telencephalic region in a sagittal section of a paraffin wax-embedded E16 embryo stained with Hematoxylin and Eosin, representing the ventricular zone (VZ), intermediate zone (IZ) and cortical plate (CP). (B) The adenovirus vector of lacZ was injected into the lateral ventricle of an E13 embryo and frozen sections of the head were prepared three days later, at E16. lacZ activity was detected by X-gal staining (light blue). The arrow indicates the accumulation of lacZ-expressing cells in the cortical plate. (C) BrdU was injected intraperitoneally into an E13-timed pregnant mouse and the embryo was fixed at E16. Paraffin wax-embedded sections of the head of the embryo were stained with anti-BrdU antibody, alkaline phosphatase-conjugated secondary antibody and BM purple. The arrow indicates the accumulation of BrdU-labeled nuclei in the cortical plate. (D) An adenovirus vector of T7-MUK was injected into the lateral ventricle of an E13 embryo and paraffin sections of the head were prepared 3 days later, at E16. Cells expressing T7-MUK were detected by immunostaining with anti-T7 antibody. The arrow indicates a cell layer accumulating T7-MUK expressing cells in the intermediate zone. (E) The adenovirus vector of T7-MUK/KR was injected and cells expressing T7-MUK/KR were detected as in D. The arrow indicates the accumulation of T7-MUK/KR expressing cells on the pial side of the cortical plate (CP). (F) A paraffin wax-embedded section prepared from a non-injected E16 embryo was immunostained as in D,E to show the background level of staining with anti-T7 antibody. (G) An adenovirus vector of T7-MUK was injected as in D, and sections were prepared at E18 for the detection of T7-MUK by immunostaining using anti-T7 antibody. The arrow indicates the remaining T7-MUK-expressing cell layer in the intermediate zone (IZ). (H) An adenovirus vector of T7-MUK was injected into the lateral ventricle of an E15 embryo and paraffin wax-embedded sections of the head were prepared three days later, at E18. The arrow indicates the T7-MUK-expressing cells accumulated in the intermediate zone (IZ). (I-K) Adenovirus vectors for T7-MUK or T7-MUK/KR were injected into the lateral ventricle of an E13 embryo. The virus-injected embryos and non-injected control embryo were fixed at E14, and paraffin wax-embedded sections were immunostained using anti-T7 antibody. Cell layers corresponding to the ventricular zone (VZ) and preplate (PP) are indicated on the left. Note the wide distribution of expression of T7-MUK and T7-MUK/KR in cells in the ventricular zone. Scale bars: 200 µm.

View larger version (135K): [in a new window]   Fig. 4. Constitutive expression of MUK does not affect the radial glial fiber architecture. Adenovirus vectors were injected into the lateral ventricle of an E13 embryo and paraffin sections of the head region were prepared 3 days later, as in Fig. 3D-F. Sections were immunostained using anti-vimentin to visualize radial glial fibers (O’Rourke et al., 1992) (red). The expression of T7-MUK or T7-MUK/KR (green) and nuclear staining with DAPI (blue) are also shown. Cortical regions of the embryos infected with adenovirus vectors for T7-MUK (A) or T7-MUK/KR (B) or non-infected control embryo (C) are shown. Note that no significant changes in radial glial fiber architecture are induced by virus infection. The big yellow spots represent blood cells stained with the secondary antibodies. Arrows indicate radial glial fibers, and arrowheads indicate cells expressing T7-MUK or T7-MUK/KR. Asterisks indicate blood vessels with nonspecific staining of erythrocyte (yellow). Layer identifications are indicated at the bottom. Note the formation of the cortical plate in T7-MUK virus infected brain, as designated by a cluster of round nuclei. Scale bar: 100 µm.

View larger version (139K): [in a new window]   Fig. 5. Comparison of endogenous and exogenous MUK-expressing cells. (A) A section of non-infected E16 embryo telencephalon was stained with anti-MUK antibody as in Fig. 1B, to represent endogenous MUK-expressing cells. Observation of the subventricular zone (SVZ) at higher magnification reveals numerous cellular processes (arrows) as well as cell bodies stained with anti-MUK antibody. (B) A section of a T7-MUK virus-injected embryo was prepared and stained as in Fig. 3D. Observation of the subventricular zone (SVZ) at higher magnification reveals numerous cellular processes (arrows), as well as cell bodies stained with anti-T7 antibody. (C) A section of non-infected E16 embryo telencephalon was stained with anti-T7 antibody to show the background staining level. (D) A section was prepared and stained as in A, except a secondary antibody conjugated with a fluorescent dye, Cy3 (red), was used to detect endogenous MUK expression. Nuclei were labeled with DAPI (blue). Immunofluorescent microscopy reveals the localization of the MUK protein at higher resolution, associated with a dot-like structure distributing close to or distant from nuclei (arrows). (E) A section of a non-infected E16 embryo was stained as in D, using anti-T7 antibody. Arrows indicate the dot-like distribution of the T7-MUK protein. (F) A section of non-infected E16 embryo was stained as in D, using anti-T7 antibody to show the background staining level. Scale bars: 20 µm.

View larger version (124K): [in a new window]   Fig. 6. Migration arrested cells show features of immature neurons. (A) A section of non-infected E16 embryonic telencephalon was stained with anti-class III ß-tubulin antibody representing postmitotic immature and mature neurons (Menezes and Luskin, 1994). Note that most cells other than ventricular cells are stained. (B) A section of T7-MUK virus injected embryo was stained with anti-class III ß-tubulin antibody. The staining profile is indistinguishable from that of the non-infected embryo (A). (C) A close section was stained with anti-T7 antibody. As a large region of subventricular zone (SVZ)/intermediate zone (IZ) cells are expressing distinct levels of T7-MUK, these cells may also be expressing anti-class III ß-tubulin. (D) The expression of anti-class III ß-tubulin in T7-MUK-expressing cells is shown by double immunostaining. Dot-like signals for T7-MUK (red) are seen on cell processes or cell bodies, representing the presence of anti-class III ß-tubulin (green), as indicated by arrows. Nuclei were labeled with DAPI (blue). (E) A section of a T7-MUK virus injected embryo was double stained using anti-T7 antibody (red) and anti-MAP2 antibody (green) to show labeling of relatively mature neurons (Menezes and Luskin, 1994). Note the heavy staining of cells in the marginal zone (MZ), cortical plate (CP) and subplate (SP), and the relatively weak staining of cells in the subventricular zone (SVZ) and intermediate zone (IZ), including T7-MUK-expressing cells (arrows). (F) A section of T7-MUK/KR virus injected embryo was double stained as in E. The staining profile of MAP2 is essentially the same as in E, and in sections of non-infected embryo (data not shown). Arrows indicate T7-MUK/KR-expressing cells. Scale bars: 50 µm.

View larger version (59K): [in a new window]   Fig. 7. MUK protein associated with microtubules and Golgi apparatus. (A) Primary culture of E16 cortical cells was stained with anti--tubulin antibody (green) and anti-MUK antibody (red). Nuclei were labeled with DAPI (blue). (B) Higher magnification of a cell prepared as in A, showing the association of the dotted structure containing MUK with microtubules. Many MUK-containing dotted structures are located at the tip of microtubules (arrows). (C) Cells were stained with anti-MUK antibody (left panel, red) and anti-GM130 antibody to visualize Golgi apparatus (middle panel, green). Merged view with nuclear staining with DAPI (blue) is also shown (right panel). Note the certain fraction of MUK localized on Golgi apparatus (arrows). Scale bars: 10 µm.

View larger version (44K): [in a new window]   Fig. 8. Overexpression of MUK modifies microtubule organization. (A) COS-1 cells were transfected with expression vectors for GFP (a,b), T7-MUK (c,d), and T7-MUK/KR (e,f). Fixed cells were double-stained using anti-T7 antibody (c,e) (green) and anti--tubulin antibody (b,d,f) (red). The expression of GFP was detected by its fluorescence (a). Arrows in c and d indicate T7-MUK-expressing cells losing the radial array of microtubules. Arrowheads indicate the radial array of microtubules observed in GFP- or T7-MUK/KR-expressing cells and cells without detectable levels of T7-MUK expression. Scale bar: 50 µm. (B) The frequency of radial array appearance was analyzed statistically. Small panels shows the definition of radial array +, ± or – cells. The appearance of the radial array is expressed as percent of the GFP-, T7-MUK-, or T7-MUK/KR-positive cells counted (n>300) (*P<0.005 versus GFP or MUK/KR control). Error bars represent the standard deviation of three independent transfection experiments.

View larger version (58K): [in a new window]   Fig. 9. Schematic drawing of radially migrating neurons in developing mouse telencephalon. Undifferentiated neuroepithelial cells located in the ventricular zone undergo asymmetrical cell division and generate postmitotic young neurons (leftmost cells). Cells leave the ventricular zone and start to differentiate and express MUK, which induces JNK activity. Simultaneously, the radial migration (vertical arrows) is retarded or pauses; this may provide a chance for tangential migration (horizontal arrows). The MUK protein level and JNK activity are reduced upon the progression of neural differentiation or by specific factors present on the pial side (upper part of this figure) of the intermediate zone. Then, a definite leading edge is formed, and radial migration is accelerated.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter