Department of Molecular Biology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan
*Author for correspondence (e-mail: sh3312{at}med.yokohama-cu.ac.jp)
Accepted 27 June 2002
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SUMMARY |
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Key words: Cortex, JNK, Microtubules, Mouse
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INTRODUCTION |
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Jun N-terminal kinases (JNKs), also known as stress-activated protein kinases (SAPKs), are members of the mitogen-activated protein kinase (MAPK) family (Kyriakis and Avruch, 2001). Double knockout of the Jnk1 and Jnk2 genes impairs neural tube closure and cortical development in the brain of mouse embryos, as well as regional specific apoptosis of undifferentiated neural stem cells (Kuan et al., 1999
; Sabapathy et al., 1999
), although the molecular mechanisms that cause these defects remain to be explored. JNK activity is regulated by protein kinases of the MAPK kinase kinase (MAPKKK) class through protein kinases of the MAPK kinase (MAPKK) class (Nishida and Gotoh, 1993
; Widmann et al., 1999
). We have identified a protein kinase related to mixed lineage kinases, MUK (also known as DLK and ZPK), as a MAPKKK of JNK (Hirai et al., 1996
) that is highly expressed in adult and embryonic neural tissues (Holzman et al., 1994
; Nadeau et al., 1997
). MUK/DLK/ZPK, as well as other mixed lineage kinases have been shown to associate with scaffold proteins, JIPs, which also interact directly with MKK7, a MAPKK class protein kinase, and JNK (Whitmarsh et al., 1998
; Ito et al., 1999
). More recently, JIPs have been identified as cargo of the kinesin motor functioning in vesicle transport (Bowman et al., 2000
; Verhey et al., 2001
). In addition, JIPs carrying MUK/DLK/ZPK also bind to a Reelin receptor, ApoER2 (Stockinger et al., 2000
; Verhey et al., 2001
). Taken together, these observations suggest that the MUK/DLK/ZPK-JNK pathway could be involved in the regulation of cellular events supported by kinesin motors and/or Reelin signaling, such as axonal transport and neural cell migration, although this possibility has not been tested.
To explore the significance of the MUK/DLK/ZPK-JNK signaling pathway in neural cell migration in vivo, we first examined the expression of the MUK/DLK/ZPK protein and the distribution of active JNK in developing mouse brain at different embryonic stages and found a temporal induction of MUK/DLK/ZPK expression and JNK activation in immature neurons. Then we monitored the effect of the constitutive activation of the MUK/DLK/ZPK-JNK pathway on neural migration and differentiation in utero, and found an inhibitory effect of MUK/DLK/ZPK expression on the radial migration of immature neurons. We also found that MUK/DLK/ZPK is associated with dotted structures located along microtubules as well as in Golgi apparatus in primary culture cells of E16 embryonic cortex, and that MUK/DLK/ZPK overexpression in COS1 cells alters microtubule organization. These results strongly suggest that the MUK/DLK/ZPK-JNK pathway contributes to the regulation of neural cell migration.
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MATERIALS AND METHODS |
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Injection of vectors into embryonic brain
The ICR mice used in these experiments were maintained according to protocols approved by the Institutional Animal Care and Use Committee at Yokohama City University School of Medicine. The uterus of an E13-timed pregnant mouse was incised under a bio-safety cabinet. Adenovirus vector (25x1010 pfu/ml) in
0.5-1 µl of PBS containing 5 mg/ml of Methyl Green was injected into the lateral ventricle of the embryonic brain using a 50 µm diameter glass micropipette connected to a 25 µl Hamilton microsyringe. After injection, 2 ml of Hanks balanced salt solution containing 0.1% glucose was added to the peritoneal cavity and the abdomen was closed.
The morning of the day on which the vaginal plug was detected was designated as E0.5.
Immunostaining of tissue sections
Embryos were fixed with 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) for paraffin wax-embedded sectioning and frozen directly in OTC compound for frozen sectioning. Hydrolyzed paraffin wax-embedded sections were heat-treated at 120°C for 20 minutes in 10 mM sodium citrate buffer, pH 6.0. For staining with anti-BrdU antibody, sections were further treated with 1 N HCl for 15 minutes at room temperature. Frozen sections were fixed with methanol/acetone 1:1 mix at 20°C for 10 minutes and air dried. To inactivate endogenous alkaline phosphatase activity, sections were treated with 3% H2O2 for 15 minutes at room temperature. Immunostaining was performed according to standard protocols using 10% normal goat serum in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) as a blocking reagent, and primary and secondary antibodies diluted in 0.1% BSA/TBST, as follows. Primary antibodies: affinity purified anti-MUK rabbit antibody raised against the C-terminal part of MUK containing 276 amino acids 1/20-1/100, omni-probe 1/300 (rabbit antibody against T7-tag, Santa Cruz), anti-class III ß-tubulin
1/50-1/200 (mouse monoclonal IgG; Chemicon), anti-MAP2 1/300 (mouse monoclonal IgG, Sigma), anti-vimentin 1/500 (mouse monoclonal IgM, Sigma), anti-active JNK/p38/MAPK (ERK) antibodies 1/1000 (rabbit, Promega), anti-
-tubulin 1/500 (mouse monoclonal IgG, Sigma), anti-GM130 1/300 (mouse monoclonal IgG, BD Transduction Lab), anti-
-tubulin 1/300 (rabbit, Sigma) and ant-active caspase3 1/200 (rabbit, Promega). Secondary antibodies: alkaline phosphatase-conjugated anti-rabbit or mouse IgG 1/2000 (TAGO), Cy3-conjugated anti-rabbit or mouse IgG 1/2000 (Amersham), Alexa488-conjugated anti-rabbit or mouse IgG (Molecular Probe) or Cy3-conjugated anti-mouse IgM 1/2000 (Jackson Immuno Res. Lab.). DAPI (2.5 µg/ml) was included in the final wash buffer (TBST) for nuclear staining.
Protein analysis
For Western blot analysis, a pair of telencephalic vesicles from an E16 embryo were cut out in ice-cold PBS, the meninges were removed, and the vesicles were homogenized in 1 ml of SDS-PAGE sample buffer. To prepare layer-specific protein samples, thick (30 µm) sections of frozen E16 embryo head were quickly dried with a blower, and three layers containing mainly the ventricular zone, intermediate zone or cortical plate were dissected with a sharp surgical blade. Cell clumps from 10-15 sections were lysed in 100 µl of sample buffer. These samples were appropriately diluted to give equal protein amounts and used for SDS-PAGE. Western blot analysis was performed according to standard protocols using the following antibodies: anti-MUK antibody 1/20, anti-vimentin antibody 1/500 (monoclonal IgM, Sigma), anti-MAP2 1/200 (monoclonal IgG, Sigma), anti-active JNK/p38/ERK antibodies 1/1000 (rabbit, Promega), anti-MAPK (ERK) 1/3000 (rabbit, Upstate Biochem) and anti-p38 1/300 (rabbit, Upstate Biochem). To detect JNKs, a mixture of anti-JNK1 1/1000 (mouse monoclonal, Phamingen) and anti-JNK2 1/2000 (mouse monoclonal, Santa Cruz) was used. For secondary antibodies, horseradish peroxidase-conjugated anti-rabbit or mouse Ig
1/2000-1/5000 (Amersham) was used; the enzyme activity was detected with an ECL plus system (Amersham) and luminescence was quantified with a FUJI Las1000 plus luminescence image analyzer. The in-gel kinase assay was performed as described elsewhere (Hirai et al., 1996
) using [
32P]ATP and Jun protein as a substrate; the JNK activity detected on the gel was quantified with a FUJI BAS200 image plate scanner system. ß-Galactosidase protein was detected in frozen sections fixed with 0.2% glutaraldehyde at room temperature for 5 minutes using X-gal solution according to the standard protocol. The section was lightly counterstained with Hematoxylin.
Cell culture and DNA transfection
NIH3T3 cells were maintained in DMEM supplemented with 7% calf serum. For UV or serum treatment, cells were starved for 36 hours with DMEM supplemented with 0.2% fetal calf serum, and then irradiated with UVC 200 J/m2 or fetal calf serum was added to a final concentration of 20%. Cells were lysed in SDS-PAGE sample buffer 40 minutes after UV irradiation or 5 minutes after serum addition. COS1 cells were maintained in DMEM supplemented with 10% fetal calf serum, and 3x106 cells were transfected with 16 µg of expression vectors for EGFP (pEGFP-c2, Clontech), T7-MUK or T7-MUK/KR by electroporation. The cells were further cultured for 24 hours and fixed with 3% PFA for 20 minutes at room temperature; immunostaining was performed as described for tissue sections. For staining with the anti--tubulin antibody, cells were fixed with methanol/acetone for 10 minutes at room temperature.
Primary cultures of neural cells were prepared from the cortical region of E16 mouse telencephalon. Cells were dispersed with 0.05% trypsin in PBS and seeded on poly-ornitin coated cover slips at a cell density of 8x104 cells/cm2. After 24 hours culture in DMEM-F12 supplemented with 5% fetal calf serum, the cells were fixed and immunostained as described above.
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RESULTS |
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JNKs are activated in cells expressing MUK
Given that MUK is an activator of the JNK pathway (Hirai et al., 1996; Fan et al., 1996
), it was predicted that JNK is activated in the intermediate zone of the developing telencephalon. In fact, we were able to detect active forms of JNKs (p46 and p55) with anti-active JNK antibody when a whole telencephalon extract was used for western blot analysis (Fig. 2A, part a). To detect the active form of JNK in situ, we examined frozen sections of E16 telencephalon by immunohistochemical staining. As shown in Fig. 2B (parts a,b), the activated JNK was detected in the intermediate zone, where the expression of MUK protein was observed. A good correspondence between the localizations of MUK and active JNK in the cortex was also observed in frozen sections of an E18 embryo at (Fig. 2B, parts f,g) and other embryonic stages (data not shown). Weak signals for activated forms of p38 and ERK were also observed in the intermediate zone and cortical plate, respectively (Fig. 2B, parts c,d), while they were barely detectable by western blot analysis (Fig. 2A, parts b,c). We confirmed the preferential expression of MUK and active JNKs in the intermediate zone by western blot analysis combined with microdissection of the three cell layers composed mainly of cortical plate, intermediate zone and ventricular zone. The protein sample in the ventricular zone was characterized by an abundance of vimentin, and that in the cortical plate was characterized by an abundance of MAP2 (Fig. 2C, parts e,f). MUK protein and active JNKs were found to be highly concentrated in the intermediate zone (Fig. 2C, parts a,b), as shown by immunohistochemical staining, while the total amounts of JNK protein and total protein as estimated by Coomassie Brilliant Blue staining did not vary very much between the fractions (Fig. 2C, parts c,d). When the ratio of the amount of active- to total-JNK was calculated from the quantitative data shown in Fig. 2C (parts b,c), the value for intermediate zone JNKs was 6.02-fold (p46 JNK, s.d.=0.98, n=3) or 15.27-fold (p55 JNK, s.d.=1.75, n=3) higher than the average value for the ventricular zone and cortical plate. We further confirmed the presence of active JNK in the intermediate zone by in-gel kinase assay (Fig. 2D). Quantification of the relative JNK activity shows 5.7-fold more activity of p46 JNK and 7.7-fold more activity of p55 JNK at the intermediate zone in comparison with the average of JNK activity at the ventricular zone and cortical plate. Taken together, these observations indicate that the MUK-JNK pathway is activated temporally in migrating immature neurons.
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To characterize the differentiation stage of the exogenous MUK-expressing cells, we examined the expression of class III ß-tubulin and MAP2. Class III ß-tubulin is expressed in most postmitotic neurons, including immature neurons located in the subventricular and intermediate zones (Menezes and Luskin, 1994) (Fig. 6A). This expression profile is also represented in the embryonic cortex, including cells expressing exogenous MUK (Fig. 6B,C), indicating the expression of class III ß-tubulin in these cells. This was confirmed by double immunofluorescent staining, showing the presence of dot-like signals for T7-MUK on the cell bodies or processes labeled with class III ß-tubulin (arrows in Fig. 6D). Taken together with the observation that postmitotic cells carrying exogenous MUK protein are not found in the ventricular zone, these observations indicate that the early step of neural differentiation is not disturbed by the ectopic expression of MUK protein.
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MUK protein localizes on microtubules and Golgi apparatus
To understand the molecular basis of MUK function in neural cell migration, we first tested the subcellular localization of MUK using primary cultures of E16 cortical cells. To avoid continual differentiation or dedifferentiation of MUK-expressing cells in culture, dispersed cells were fixed after 24 hours of culture. As shown with tissue sections of cortex (Fig. 5D), the MUK protein associates with different sized dotted structures in primary culture cells (Fig. 7A). When merged with tubulin staining, it appeared that most of these dotted structures were located along microtubules, often at the tips of microtubules (Fig. 7B). MUK also associates with the Golgi apparatus located in the perinuclear region (Fig. 7C), as reported for NIH3T3 cells (Douziech et al., 1999).
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DISCUSSION |
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In combination with our previous report that MUK/DLK/ZPK is a MAPKKK for the JNK pathway (Hirai et al., 1996; Fan et al., 1996
), the coincidence of MUK/DLK/ZPK expression and JNK activation in the developing cortex indicates that the JNK activity is regulated by the expression level of MUK/DLK/ZPK. The preferential activation of JNK among three MAPK-related protein kinases in the E16 telencephalon is consistent with observations in COS cells overexpressing MUK/DLK/ZPK (Hirai et al., 1997
; Merritt et al., 1999
). The ratio of active JNK to total JNK in the intermediate zone of the E16 cortex is roughly comparable with that in NIH3T3 cells irradiated with UV, a potent activator of JNK (see Fig. 2A, part a; Fig.2C, part b; Fig. 2D), indicating the potency of JNK activity in differentiating neurons. The artificial activation of JNK during the course of sample preparation is not likely to have occurred, because samples for frozen sections and micro dissection were prepared by quick freezing in liquid nitrogen within one minute of sacrificing by cervical dislocation (see Materials and Methods). In addition, no significant changes in JNK activity were observed in E16 brain samples before or after incubation in ice-cold PBS for 30 minutes (S. H., unpublished).
How the expression of MUK/DLK/ZPK is controlled is not clear as yet. Even though the MUK/DLK/ZPK protein level is downregulated in the cortical plate of the E16 embryonic telencephalon, the mRNA level, as estimated by in situ hybridization, is still high (Nadeau et al., 1997) (S. H., unpublished). Therefore, translational or post-translational regulation in the cortical plate may be responsible for the suppression of the MUK/DLK/ZPK protein level. At a late embryonic stage, E18, the MUK/DLK/ZPK protein begins to be found on the ventricle side of the cortical plate, and it is widely expressed in all differentiated neurons in the neocortex postnatally. The MUK/DLK/ZPK-JNK pathway may therefore function not only in a temporal event in the early stages of neural differentiation, but also in general microtubule-dependent neuronal events, such as axon outgrowth and axonal transport.
Given that the MUK/DLK/ZPK-JNK pathway induces apoptosis in cultured neural cells (Xu et al., 2001), it is predicted that apoptosis is predominantly induced when cells cross the intermediate zone. However, it was difficult to detect cells meeting certain criteria for apoptosis, nuclear condensation and the expression of the active form of caspase 3, in the intermediate zone and cortical plate. Moreover, no layer-specific increase in the percentage of dying cells as detected by the in situ end-labeling method was observed in the telencephalon of normal E16 embryos (Blaschke et al., 1996
). Therefore, the MUK/DLK/ZPK-JNK pathway might not induce apoptosis in the developing telencephalon.
The effect of exogenous MUK/DLK/ZPK expression is cell-autonomous, and the radial glial scaffold and radial migration of other neurons are apparently not affected. Therefore, MUK/DLK/ZPK expression may affect the interaction with radial glia and/or cell migration itself. It should also be noted that the MUK/DLK/ZPK protein associates with dotted structures that are frequently located along microtubules and that the overexpression of MUK/DLK/ZPK in COS-1 cells induces microtubule reorganization, as characterized by the disappearance of radial microtubule organization without massive depolymerization of the microtubules and disruption of centrosomes. As a well-organized microtubule network is essential for the directed migration of cells (Gotlieb et al., 1981; DeRouvroit and Goffinet, 2001
), these observations may explain, at least in part, the neuronal migration disorder observed with the constitutive expression of MUK/DLK/ZPK. This unique type of microtubule disorganization has not been commonly reported except in the case of Lis1 overexpression in COS-7 cells (Smith et al., 2000
). The LIS1 gene is responsible for a neural cell migration disorder and type I lissencephaly in humans; the Lis1 protein associates with the dynein-dynactin complex, a microtubule motor (Smith et al., 2000
; Faulkner et al., 2000
; Liu et al., 2000
). Therefore, the similar effects of MUK/DLK/ZPK and Lis1 overexpression on microtubule organization support the idea that the MUK/DLK/ZPK-JNK pathway regulates neural cell migration via microtubule-based event.
Genetic arguments about the function of JIPs, which are scaffold proteins for the MUK/DLK/ZPK-JNK pathway, also support this notion. Sunday driver, a Drosophilla homolog of JIP3/JSAP1, has recently been identified as a receptor for kinesin motors and to function in vesicle transport (Bowman et al., 2000; Verhey et al., 2001
). Moreover, Caenorhabditis elegans JNK and JNK kinases, as well as UNC-16, a nematode homolog of JIP3, have been shown to regulate vesicle transport in neurons (Byrd et al., 2001
). As vesicle transport supports cell locomotion by driving the endocytic cycle (Brestscher, 1984
), JIP and JNK may be essential not only for axonal transport but also for neural cell migration. The MUK-associated dotted structures observed in the primary culture of cortical cells may correspond to vesicular cargo in secretory or endocytic pathways. Notably, mammalian JIPs bind to a Reelin receptor, ApoER2, as well as to the kinesin light chain (Stockinger et al., 2000
; Verhey et al., 2001
). Therefore, they may mediate the transport of vesicular cargoes containing the Reelin receptor, which is essential for the radial migration of neural cells in the cortical plate (DArcangelo et al., 1995
; Trommsdorff et al., 1999
; Senzaki et al., 1999
). Even though this possibility has not been tested, our results suggest the intriguing possibility that the MUK/DLK/ZPK-JNK pathway supported by JIPs transfers a signal from Reelin to microtubules for the regulation of neural cell migration.
Although the constitutive expression of exogenous MUK/DLK/ZPK driven by the CAG promoter arrests the radial migration of neural cells, the expression of the endogenous MUK/DLK/ZPK gene does not cause such a migration disorder. This difference might be explained by the temporal features of endogenous MUK/DLK/ZPK expression, that is, the total downregulation of MUK expression on the cortical plate side of the intermediate zone. In that case, it is interesting to know whether the radially directed migration of normal immature neurons is interrupted at the intermediate zone where high levels of endogenous MUK/DLK/ZPK expression and JNK activation are observed. It has been reported that a single neural cell changes the velocity of radial migration depending on its position (ORourke et al., 1992; Nadarajah et al., 2001
). In addition, cells in the intermediate zone often have numerous processes, rather than the single definite leading process observed in neural cells migrating in vitro or in slice cultures of neonatal ferret brain (ORourke et al., 1992
; Rivas and Hatten, 1995
). Cells with a definite leading process are found on the cortical plate side of the intermediate zone (Shoukimas and Hinds, 1978
), where MUK/DLK/ZPK expression and JNK activity are significantly downregulated. Such sequential cell-shape changes of migratory neurons have been clearly shown by retrovirus vector-mediated GFP labeling of neurons in E15-E18 rat embryonic telencephalon (Noctor et al., 2001
). Numerous randomly oriented cell processes are also visible by optical microscopy in MUK/DLK/ZPK expressing cells (Fig. 5A). Although the relationship between the activation of the MUK/DLK/ZPK-JNK pathway and the migration rate is not clear at present, these observations, together with the results of MUK overexpression, suggest that MUK/DLK/ZPK-JNK modulates microtubule organization and temporally disrupts the unipolar cell shape, and probably also the radial migration of immature neurons just after they leave ventricular zone (Fig. 9). This step may be required for the newly generated neurons to prepare for maturation and provide them with a chance for tangential dispersion, which is also essential for the formation of a functional neocortex (Rakic, 1990
; ORourke et al., 1992
; Parnavelas, 2000
).
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ACKNOWLEDGMENTS |
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