A key role for Pak4 in proliferation and differentiation of neural progenitor cells

Developmental Biology 353 (2011) 206–216

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Developmental Biology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / d e v e l o p m e n t a l b i o l o g y

A key role for Pak4 in proliferation and differentiation of neural progenitor cells Yanmei Tian, Liang Lei, Audrey Minden ⁎ Department of Chemical Biology, Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854, USA

a r t i c l e

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Article history: Received for publication 16 June 2010 Revised 31 January 2011 Accepted 15 February 2011 Available online 4 March 2011 Keywords: Pak4 Neurogenesis Conditional knockout Nestin Flox

a b s t r a c t The Pak4 serine/threonine kinase regulates cytoskeletal organization, and controls cell growth, proliferation, and survival. Deletion of Pak4 in mice results in embryonic lethality prior to embryonic day 11.5. Pak4 knockout embryos exhibit abnormalities in the nervous system, the heart, and other tissues. In this study a conditional deletion of Pak4 was generated in order to study the function of Pak4 in the development of the brain. Nervous system-specific conditional deletion of Pak4 was accomplished by crossing mice with a floxed allele of Pak4 with transgenic mice expressing Cre recombinase under the control of the nestin promoter. The conditional Pak4 knockout mice were born normally, but displayed growth retardation and died prematurely. The brains showed a dramatic decrease in proliferation of cortical and striatal neuronal progenitor cells. In vitro analyses revealed a reduced proliferation and self-renewing capacity of neural progenitor cells isolated from Pak4 knockout brains. The mice also exhibited cortical thinning, impaired neurogenesis and loss of neuroepithelial adherens junctions. By the time the mice died, by 4 weeks after birth, severe hydrocephalus could also be seen. These results suggest that Pak4 plays a critical role in the regulation of neural progenitor cell proliferation and in establishing the foundation for development of the adult brain. © 2011 Elsevier Inc. All rights reserved.

Introduction Pak4 was originally identified as a protein which mediates Cdc42 induced filopodia formation (Abo et al., 1998). Along with Pak5 and Pak6, Pak4 is a member of the group B family of Pak serine/threonine kinases (Abo et al., 1998; Jaffer and Chernoff, 2002). The group A family includes Paks 1, 2, and 3. Pak2 and Pak4 are ubiquitously expressed in different tissues, whereas Paks 1, 3, 5 and 6 have more limited tissue-specific expression patterns (Bagrodia and Cerione, 1999; Daniels and Bokoch, 1999). In addition to its function in cytoskeletal organization, Pak4 has been reported to be involved in multiple signaling pathways, including oncogene induced premature senescence and cell survival (Cammarano et al., 2005; Gnesutta and Minden, 2003; Gnesutta et al., 2001; Li and Minden, 2005; Lu et al., 2003). Mouse knockouts of the Paks have revealed critical roles for these proteins in the nervous system. Pak3 knockout mice exhibit abnormal synaptic plasticity and have learning and memory defects (Meng et al., 2005). Pak5 and Pak6 knockout mice appear phenotypically normal (Li and Minden, 2003; Nekrasova et al., 2008), but Pak5/Pak6 double knockout mice have locomotor and learning deficits (Nekrasova et al., 2008). In sharp contrast to Pak3, Pak5 and Pak6, Pak4 knockout mice are embryonic lethal, and die by

⁎ Corresponding author. Fax: +1 732 445 0687. E-mail address: [email protected] (A. Minden). 0012-1606/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2011.02.026

embryonic day 11.5 (E11.5). Pak4-null embryos have a number of abnormalities, including abnormalities in the heart, and defects in the nervous system (Qu et al., 2003). In the Pak4 null embryos, spinal cord motor neurons and interneurons fail to differentiate and to migrate to their proper locations. Throughout the brain, the mice also display a lack of axonal outgrowth, and an abnormally thin neuroepithelium. Important roles for Pak proteins in the nervous system were also noted by other studies. Drosophila PAK (DPAK), a group A Pak, has been shown to be necessary for photoreceptor axon guidance (Hing et al., 1999), and Mushroom Body Tiny (MBT), a group B Pak, may regulate neuronal survival or proliferation (Melzig et al., 1998). In humans, mutations in PAK3 have been linked to nonsyndromatic X-linked mental retardation (Allen et al., 1998). In mammalian cells, expression of PAK1 triggers neurite outgrowth in PC12 cells, and activated PAK5 promotes filopodia formation and neurite outgrowth in N1E-115 neuroblastoma cells (Dan et al., 2002). The neuronal abnormalities in the Pak4 knockout mice are striking (Qu et al., 2003), but the information that can be obtained from studying these knockout mice is limited by the early embryonic lethality. In this study, we explored the role of Pak4 in the development of the brain more directly, by generating conditional knockout mice in which Pak4 is specifically deleted in the central nervous system (CNS). This involved crossing mice with a floxed allele of Pak4, with mice containing nestin-Cre (Tronche et al., 1999), where the Cre recombinase is expressed in the nervous system. The resulting nestin-Cre-mediated conditional Pak4 knockout mice appeared

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normal at birth but died prematurely. A dramatic decrease in proliferation of neural progenitor cells was evident in the knockout embryos and newborns, as well as abnormal neurogenesis. Our results indicate that there is a functional requirement for Pak4 in proliferation of neural progenitor cells and in cortical neurogenesis.

and the proportion of BrdU+/Ki67− cells (cells withdrawn from cell cycle) to total BrdU+ cells was calculated.

Materials and methods

Neurons were isolated from cortices of E14.5 mouse embryos as previously described (Nekrasova et al., 2008). The dissociated cortical cells were plated onto 8-well chamber slides coated with poly-L-lysine (Sigma) in serum-free Neurobasal medium (Gibco) containing B-27 and N-2 supplements (Invitrogen) and penicillin/ streptomycin. The plating density was 3000 cells/cm2 and 30,000 cells/cm2. Cells were incubated in a CO2 incubator at 37 °C, and half of the media was changed every 2 days. For immunocytochemistry, cells were fixed with 4% paraformaldehyde, permeabilized with icecold methanol, and subjected to indirect immunostaining with antiβ-tubulin III and FITC-conjugated secondary antibody. Cells were also counter-stained with DAPI. Neurites were defined as processes developed directly on the cell body (soma) of neuron. Number of total branches was defined as number of total protruding tips from a neuron. Number of branches on longest neurite was defined as number of total protruding tips extended from the longest neurite on a cell. For neurosphere assays, dissociated cortical cells from E14.5 embryos were suspended in serum-free Neurobasal medium containing B-27 and N-2 supplements, basic fibroblast growth factor (bFGF, 20 ng/ml), epidermal growth factor (EGF, 20 ng/ml) (Invitrogen), and penicillin/streptomycin. Single cells were then seeded onto uncoated 8-well chamber slides at low density (3000 cells/cm2) and incubated in a CO2 incubator at 37 °C. Neurospheres were observed under an optical microscope. Pictures were taken at the same resolution and the pixel number for the area of single neurosphere was measured with Adobe Photoshop. Only the ones with pixel numbers greater than 300 were counted and analyzed.

Mice Generation of Pak4 floxed mice (Pak4f/f) and Pak4 deletion mice (Pak4+/Δ) were previously described (Tian et al., 2009). For neuralspecific deletion, female Pak4+/Δ mice were first crossed to nestinCre mice (obtained from Jackson Laboratory) to generate the breeding male nestin-Cre;Pak4+/Δ. Such breeding males were then bred to Pak4f/f female mice. In timed mating, noon of the vaginal plug detection date was termed day 0.5 of gestation (E0.5). Brain lysate and Western blotting Brain tissues were lysed in lysis buffer containing protease inhibitors (1 mM PMSF, 10 μg/ml leupeptin and 10 μg/ml aprotinin). Brain lysates were then analyzed by Western blotting. Primary antibodies used were anti-Pak4 (Cell Signaling), anti-β-tubulin III and anti-actin (Sigma). Histology and immunohistochemistry Freshly dissected brains were fixed in 4% paraformaldehyde. For histology, fixed brains were embedded in paraffin, cut into 5–10 μm sections and stained with hematoxylin and eosin according to a standard protocol. For immunohistochemistry, fixed brains were frozen in OCT medium and cut into 20 μm sections. The cryosections were immunostained with Ki67 (DakoCytomation) or calretinin (Chemicon) antibodies using a standard avidin–biotin peroxidase complex method (Liu et al., 2008). Sections were lightly counterstained with eosin to show the structure of brains. TUNEL assay and immunofluorescence Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed with ApopTag red in situ apoptosis detection kit (Chemicon). For immunofluorescence staining, brains were fixed in 4% paraformaldehyde, frozen in OCT medium and cut into 10–20 μm sections. The cryosections were subjected to indirect immunostaining and visualized with Nikon fluorescent microscope. Primary antibodies used were anti-CDP, anti-Brn1 (Santa Cruz), anti-Foxp2, anti-Ki67, anti-Nestin (Abcam), anti-β-tubulin III, antiβ-catenin, anti-BrdU (Sigma) and anti-PH3 (Phospho-Histone H3 (Ser10)) (Cell Signaling). FITC- or TRITC-conjugated second antibodies were from Jackson ImmunoResearch Laboratories. BrdU labeling Analysis of cell cycle length and cell cycle exit was performed as described (Chenn and Walsh, 2002). Briefly, a single dose of BrdU (50 μg per kg body weight) was injected intraperitoneally into pregnant females at E17.5. After 30 min (for cell cycle length analysis) or 24 h (for cell cycle exit analysis), brains were removed from embryos, fixed in paraformaldehyde, and cryosectioned. Sections were stained with BrdU and Ki67 antibodies. For the cell cycle length assay, Ki67-positive (Ki67+) cells and Ki67 and BrdU-double positive (Ki67+/BrdU+) cells were counted and the proportion of Ki67+/ BrdU+ cells to total Ki67+ cells was calculated. For cell cycle exit analysis, BrdU+/Ki67+ cells and BrdU+/Ki67− cells were counted

Culture of dissociated neurons, immunocytochemistry and neurosphere assay

Statistical analysis Data were expressed as the mean ± SEM. Unpaired t test was performed with GraphPad Prism software (GraphPad, San Diego, CA) and the difference was considered significant if the two-tailed p value is b0.01 or 0.05. Results Disruption of Pak4 in the CNS leads to postnatal lethality and hydrocephalus Previous studies suggest that Pak4 is broadly expressed in adult tissues and in embryos as early as day 7 (Qu et al., 2003). In order to obtain mice in which Pak4 is specifically deleted in the nervous system, Pak4Flox mice were crossed with transgenic mice that express Cre under the nervous system-specific enhancer from the nestin gene, thus directing Cre expression to the nervous system. Nestin-Cre is expressed in the neuroectoderm before E9.5. By E12.5, a nestin reporter gene shows brain and spinal cord specific expression (Haigh et al., 2003). Nestin-Cre;Pak4f/Δ (Pak4cKO) mice were born at the expected Mendelian ratio and their external appearance was indistinguishable from control littermates at birth. However, all conditional knockout mice died between P13 and P31, except for two mice that reached P40 and P46 (Fig. 1A). Western blotting confirmed that Pak4 is effectively deleted in the brains of Pak4cKO mice (Fig. 1B). Prior to death, all mutant mice exhibited growth retardation, dome-like heads, and a hunchback posture (Fig. 1C). Brains from Pak4cKO mice were abnormally enlarged with excessive fluid, indicating hydrocephalus (Fig. 1D). As the animal grew, hydrocephalus became worse and the brain tissue was gradually

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Fig. 1. Disruption of Pak4 in the CNS leads to postnatal lethality due to hydrocephalus. (A) Survival curve showing the percentage of Pak4cKO mice that survived to various time points after birth. A total of 94 mice were observed. The majority of Pak4cKO mice died between 2 and 4 weeks after birth. (B) Western blot showing that Pak4 protein (arrow) is completely missing in the Pak4cKO brains. (C) Pictures of 4-week-old Pak4cKO mouse and control mouse. Pak4cKO mice were growth retarded and developed dome-shaped heads and hunchback postures prior to death. The Pak4cKO mouse died 4 days later. (D) Brains from 3-week-old control and mutant mice. The forebrains of Pak4cKO mice were highly enlarged with excessive fluid inside, indicating hydrocephalus. (E) Histological analysis showing hydrocephalus in Pak4 mutant brains. Upper panel: coronal sections of brains from P0 littermates were stained with hematoxylin/eosin (HE). Enlarged lateral ventricles and disruption of the septum (arrow) are observed in Pak4cKO brains. Lower panel shows severe hydrocephalus in 2-week-old Pak4cKO mouse. Scale bars: 1 cm in C, D; 1 mm in E.

lost. Coronal sections revealed dilated lateral ventricles with a disruption of the septum, which was evident in as early as P0 (Fig. 1E). Hydrocephalus is usually due to blockage of cerebrospinal fluid (CSF) flow and/or overproduction of CSF. This can occur in response to various brain abnormalities such as genetic defects, hemorrhage into the brain, meningitis, tumors or head injury (Crews et al., 2004). Although damage to the brain in response to severe hydrocephalous is probably the ultimate cause of death in the Pak4cKO mice, we think it is likely that the hydrocephalous is actually a secondary defect that occurs in response to other abnormalities that are triggered by the absence of Pak4. To address what are some of the more direct results of Pak4 deletion in the brain, we examined the brains of Pak4cKO mice at different ages. Pak4 mutant mice display thinning of the cerebral cortex Histological analysis revealed that the cortices of the Pak4cKO mice were reduced in thickness, which was obvious at as early as P0 (Fig. 2A). To determine whether the cortical thinning was layer-specific or a generalized reduction in total cortical thickness, the expression patterns of cortical layer-specific markers were examined. At P4, cortical layer I, identified by calretinin staining, was unchanged in the Pak4cKO brains (Fig. 2B). Layers II–III identified by either CDP or Brn1 staining were reduced by 70% compared with the brains of control mice (Fig. 2C, D). Layer IV, stained with Brn1, was reduced by 75% compared to the control brains (Fig. 2D). Layer VI, stained with Foxp2, was reduced by 84% of the control (Fig. 2E). Thus, while there was thinning in all of the cortical layers, the later born upper layers were more dramatically affected than the earlier born deeper layers and layer I.

were indistinguishable in position, thickness and brightness (Fig. 3A). At E16.5, the signal (brightness) of β-tubulin III staining was significantly reduced in the cortices of the Pak4cKO brains, compared to control mice (Fig. 3B). To gain insight into potential mechanisms behind the impaired neurogenesis in Pak4cKO brains, we isolated and cultured cortical neurons from control and mutant mice at E14.5. The neurites and branches were observed and measured. Neurites were defined as processes developed directly on the cell body (soma) of neuron. At day 5 in vitro (div5), in control cultures, ~30% of neurons developed a straight long (N400 μm) neurite that had very few (≤4) branches on it (Fig. 3C(i)). However, neurons with such a long neurite that contained few (≤4) branches on it were totally missing in the Pak4cKO cultures. As shown in Fig. 3C(ii), the number of neurites per cell in Pak4cKO cultures was comparable to control cultures, indicating that formation of the initial neuronal processes from the cell body (soma) was not affected by Pak4 deletion. However, the number of total branches per cell and the number of branches that branch from only the longest neurite of each individual cell were both significantly higher in the Pak4cKO cultures than in the control cultures. In contrast to the increased branching, the length of the longest neurite per individual cell was significantly reduced in the Pak4cKO cultures (Fig. 3C(iii)). Taken together, the Pak4cKO cells tended to have shorter neurites and more branches. When higher density cultures were examined, the Pak4cKO cultures showed an overall decrease of β-tubulin III staining (Fig. 3D). This is consistent with the results from β-tubulin III staining of brain sections (Fig. 3B). Changes in the length and branching of neurites in the absence of Pak4 may potentially account for the impaired neurogenesis found in Pak4cKO cortices and high density cultures.

Cortical neurogenesis is impaired in Pak4 mutant brains To determine whether the lack of Pak4 alters neuronal cell differentiation/migration in the cortex, we examined expression of β-tubulin III, a pan-neuronal marker. At embryonic day 14 (E14.5), the patterns of β-tubulin III staining in the wild-type and knockout mice

Pak4 is required for proliferation of neural progenitor cells in vivo and in vitro Cortical thinning and impaired neurogenesis can result from changes in cell proliferation and cell death. Pak4 has been shown to

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Fig. 2. Cortical thinning in Pak4cKO brains. (A) HE-stained coronal sections of the cerebral cortices of P0 mice. Compared to the control cortex, the Pak4cKO cortex was reduced in thickness and showed a less organized structure. (B–E) Immunostaining with calretinin (B), CDP (C), Brn1 (D) and Foxp2 (E) antibodies showing cortical layers in P4 mice. Scale bars: 100 μm. (F) The thickness of each layer was measured. Significant differences between control and Pak4cKO brains were found in layers II–III (**P b 0.01), IV (**P b 0.01) and VI (*P b 0.05).

have an important role in cell survival and cell growth (Abo et al., 1998; Gnesutta and Minden, 2003; Gnesutta et al., 2001; Li and Minden, 2005; Liu et al., 2008; Qu et al., 2001). Brain sections were therefore examined with markers for proliferation and survival. Proliferation was detected by staining with the proliferation marker Ki67. Here we found significant differences between the control and Pak4cKO brains (Fig. 4A). At all stages, proliferating cells were seen mainly in the neural progenitor cells around lateral ventricles. In the cortex, the amount of proliferating cells in both control and Pak4cKO brains appeared to be highest at the earliest time point, E14.5. As the animals developed, the amount of proliferating cells in the control brains was reduced. Interestingly, however, the amount of proliferating cells dropped off much more quickly in the Pak4cKO brains. At E14.5, the thickness of the main Ki67-positive region (ventricular zone) in the mutant cortices was comparable to those of the control brains (Fig. 4A(i, iii)). At E16.5–E18.5, in both control and Pak4cKO cortices, the proliferative cells were now detected in both the ventricular zone and the intermediate zone. The Ki67-positive layer in the subventricular and ventricular zones, however, was reduced in thickness and had fewer proliferating cells in the Pak4cKO cortices, compared to the controls (Fig. 4A(i, iii, iv)). At E18.5, the difference was more striking at the ventricular zone. In control brains, a number of Ki67-positive cells were densely lined along lateral ventricle, while in Pak4cKO brains, the density of Ki67-

positive cells lining lateral ventricle was dramatically reduced (Fig. 4A (i)). By P0, the amount of proliferating cells was quite low in cortices from control brains. The amount of proliferating cells was dramatically lower, however, in the Pak4cKO brains, compared with the control brains (Fig. 4A(i, iii, iv)). Interestingly, in the lateral walls of the lateral ventricles facing the striatum, the differences in proliferation were even more dramatic. In this subventricular zone there were a large number of proliferating cells at P0 in the control brains. In sharp contrast, very few proliferating cells could be seen in the corresponding region of the Pak4cKO brains (Fig. 4A(i, ii, iv)). These results suggest that although neural progenitors in normal brains naturally lose their capacity to proliferate as the embryos progress through development, the loss of the proliferation capacity of the neural progenitors is expedited when Pak4 is deleted. The lack of proliferation was particularly dramatic in the subventricular zone located between lateral ventricle and striatum, which interestingly is also a site of adult neurogenesis. In contrast to proliferating cells, there was no significant difference between the control and Pak4cKO brains when the amount of apoptotic cells was examined, using the TUNEL assay (Fig. 4B(i, ii)). The results above suggest that a major difference between the control and Pak4cKO brains may be the decreased level of proliferation of neural progenitor cells. We therefore further investigated the proliferation potential of neural progenitor cells in vitro. We isolated

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Fig. 3. Neurogenesis was impaired in the Pak4cKO cortex. (A, B) Coronal sections of E14.5 (A) and E16.5 (B) cortices from control and Pak4cKO mice were immunostained with β-tubulin III antibody (green). (A) At E14.5, the staining in the Pak4cKO cortex was comparable with the control cortex. (B) Neurogenesis was impaired in the Pak4cKO cortex at E16.5. (B(i)) The upper panel shows a low magnification view. In control brains, the β-tubulin III signal was strongest in outer cortex (see bracket) and in a narrow zone (indicated with an arrow) in the middle of cortex. The signals in those two zones were both reduced in Pak4cKO brains. The lower panel shows the cortices at higher magnifications. The β-tubulin III signal was significantly reduced in outer and middle parts of Pak4cKO cortices (both of which are indicated by brackets), compared to control brains. LV, lateral ventricle. (B(ii)) Western blotting confirmed the reduced expression level of β-tubulin III in the Pak4cKO brains. (C, D) Neuronal process in vitro. E14.5 cortical cells were dissociated and plated on poly-L-lysine coated chamber slides. At div5, cells were stained with β-tubulin III antibody (green) and DAPI (blue). (C) Low density cell culture. Neurons that has a straight long (N 400 μm) neurite with fewer (≤ 4) branches on it (arrows point to the cell bodies of such neurons in the control cultures) were totally missing in the Pak4cKO cultures. (C(ii)) The number of neurites, total branches, and branches on the longest neurite per individual neuron was counted. No significant difference between control and Pak4cKO cultures (P N 0.05) was found in the number of neurites per neuron. However, the number of total branches per cell, and the number of branches on the longest neurite per cell were significantly increased in Pak4cKO cultures, compared to control cultures (*P b 0.05). (C(iii)) The lengths of the longest neurite for individual cell were measured. Neurons in the control cultures developed significantly longer neurites than the neurons in Pak4cKO cultures (*P b 0.05). (D) Seeding at higher cell density. Compared to the control culture, the Pak4cKO culture showed overall weaker staining for β-tubulin III. Scale bars: 100 μm.

neural cells from the cerebral cortex at E14.5 and cultured them in the presence of bFGF and EGF. The neural progenitor cells undergo continued cell division and form free-floating spherical clusters of cells, referred to as neurospheres. The number and size of the neurospheres that form reflect the proliferation and self-renewal capacity of neural progenitor cells. As shown in Fig. 4C(i, ii, iii), the number and size of neurospheres derived from Pak4cKO cortex were significantly reduced, compared to those derived from control embryos, indicating an impaired proliferation capacity when Pak4 is deleted. These results are consistent with the defective proliferation found in Pak4cKO brain sections, suggesting that Pak4 is required for the proliferation of neural progenitor cells. The number of neural progenitor cells in the ventricular zone was decreased in the Pak4cKO mice To determine whether decreased proliferation (Fig. 4) changes the number of neural progenitor cells, brain sections from control and Pak4cKO mice were stained with nestin antibody (Fig. 5). At E18.5, significant change was found in the ventricular zone of the mutant brain. In control brain, nestin signal was detected in the ventricular zone and the intermediate zone, but the signal was much stronger in the ventricular zone. Compared to control brain, the nestin signal in

the ventricular zone of Pak4cKO brain was significantly reduced. Costaining with nestin and Ki67 showed that similar to the nestin signal, the proliferating (Ki67-positive) cells were also decreased in the ventricular zone of Pak4cKO brains compared to the controls, suggesting that the reduced number of neural progenitors may be closely related to the decreased proliferation level. Normal cell cycle in Pak4cKO brains The mechanism behind the decreased cell proliferation level in the Pak4 knockout brains is unknown. Decreased mitotic rate or failure of cell cycle re-entry may result in reduction of proliferation level. To reveal potential changes in the cell cycle, pregnant mice were injected with BrdU at E17.5 and embryonic brains were collected after 30 min or 24 h. Brain sections were stained with BrdU and Ki67. For cell cycle length, the proportion of proliferating (Ki67-positive) cells labeled by a 30-min pulse of BrdU was counted (Fig. 6A). Because in mammalian cells the length of S phase remains relatively constant while the length of G1 regulates proliferation (DiSalvo et al., 1995), the ratio between the BrdU-positive and Ki67positive cells provides an estimation of the cell cycle length. If the cell cycle lengthens, the proportion of BrdU-positive cells to total proliferating cells will decrease. Quantitation of the results indicated

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that there was no significant change in the cell cycle length in the Pak4cKO brains (Fig. 6A(ii)). Next we examined the cell cycle withdrawal in embryonic brains collected 24 h after BrdU injection (Fig. 6B). Cells that had exited the cell cycle were identified as BrdU-positive and Ki67negative. The fraction of cells exiting cell cycle in total BrdUpositive cells was not significantly changed in the Pak4cKO brains (Fig. 6B(ii)). Taken together, the reduced proliferation level in the Pak4cKO brains was not due to changes in cell cycle length or cell cycle withdrawal.

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Decreased mitosis at the ventricular surface of Pak4cKO brains To reveal potential changes in cell division, embryo brain sections were immunostained for phospho-histone H3 (PH3), which is present in late G2/M phase of the cell cycle. As shown in Fig. 7A, there were very few dividing (PH3-positive) cells at the ventricular surface of cortices in Pak4cKO brains at E18.5, while in control brains, a number of dividing cells could be seen along ventricular surface. The significant difference in cell division at the ventricular surface started to be seen at E16.5 (Fig. 7B). There is no significant difference in the

Fig. 4. Disruption of Pak4 significantly reduced proliferation of neural progenitor cells. (A) Ki67 immunostaining of coronal sections from control and Pak4cKO brains revealed decreased proliferation of neural progenitor cells in the Pak4 mutant brain. (A(i)) The right two panels are high magnifications of the boxed areas in the left two panels. Arrows indicate Ki67-positive cells. LV, lateral ventricle; ST, striatum; VZ, ventricular zone; SVZ, subventricular zone. Scale bars: 200 μm in left two panels; 50 μm in right two panels. (A(ii)) High magnifications of the dash-boxed areas in P0 left two panels in A(1) showing the striatal subventricular zone. Scale bar: 50 μm. (A(iii)) The thicknesses of VZ/SVZ and/or Ki67-positive zones were measured. The VZ/SVZ in E16.5 and the whole Ki67-positive zone in E18.5 were significantly reduced in thickness in Pak4cKO brains. **P b 0.01. (A(iv)) The number of Ki67-positive cells per field in the E16.5-P0 cortical VZ/SVZ and in the P0 striatal SVZ was counted. The density of Ki67-positive cells was significantly reduced in Pak4cKO brains at E18.5 and P0. **P b 0.01. (B) TUNEL assays of sections from control and Pak4cKO brains. Scale bar: 100 μm. (B(ii)) The number of apoptotic cells per field in the outer and the inner cortex was counted. No difference in apoptosis was seen between control and Pak4cKO cortices. P N 0.05. (C) Proliferation of neural progenitor cells in vitro. Cells were isolated from cerebral cortices at E14.5 and cultured in the presence of bFGF and EGF. Neurospheres were observed 10 days after initial seeding. Scale bar: 500 μm. Neurospheres formed in Pak4cKO culture were significantly reduced in number (c(ii), number of neurospheres per 1000 cells, **P b 0.01) and in size (C(iii), pixel number for individual neurosphere area on pictures, *P b 0.05) as compared to the controls.

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Fig. 4 (continued).

Fig. 5. Reduced number of neural progenitor cells in the ventricular zone of the Pak4cKO brain. Sections of control and Pak4cKO brains at E18.5 were co-stained with nestin (red; stains neural progenitor cells) and Ki67 (green) antibodies. Major differences in nestin staining were seen in the ventricular zones (VZ, indicated by brackets) of control and Pak4cKO brains. In control brain, nestin staining was strong in the VZ, especially at the edge of the cortex facing the lateral ventricle, indicating that the density of neural progenitor cells was high in this area. Compared to control, nestin staining was significantly reduced in the VZ of Pak4cKO brain. Ki67 staining for proliferating cells showed similar results. Compared to the control, proliferating cells in the VZ of Pak4cKO brains were significantly decreased. Scale bar: 100 μm.

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Fig. 6. Normal cell cycle in Pak4cKO brains. Pregnant mice were injected with BrdU at E17.5 and embryos were collected after 30 min for cell cycle length analysis, or after 24 h for cell cycle exit assays. Brain sections were co-stained with BrdU (red) and Ki67 (green) antibodies. Scale bars: 100 μm. (A) 30-min pulse of BrdU. In these sections, Ki67 and BrdU double positive (Ki67+/BrdU+) cells and total proliferating (Ki67+) cells were counted. Relative cell cycle length was estimated by the ratio between Ki67+/BrdU+ cells to total Ki67+ cells. (A(ii)) The percentage of Ki67+/BrdU+ cells to total Ki67+ cells was not significantly altered in Pak4cKO brains (P = 0.8). (B) 24 h after a single BrdU pulse. In these sections, BrdU+/Ki67− cells (cells withdrawn from the cell cycle) and total BrdU + cells were counted. (B(ii)) The percentage of cells exiting cell cycle to total BrdU+ cells was not significantly altered in Pak4cKO brains (P = 0.5).

number of dividing cells in cortices away from the ventricular surface at these stages. Loss of neuroepithelial adherens junctions in Pak4 mutant brains Stainings for proliferation (Fig. 5) and cell division (Fig. 7A) showed that the ventricular surface is the area most dramatically affected by Pak4 deletion in the brain. At the ventricular interface, the apical tips of neuroepithelial/neural progenitor cells are connected by adherens junctions. Abnormalities in adherens junctions cause hydrocephalus and postnatal death (Imai et al., 2006; Klezovitch et al., 2004; Ohtoshi, 2008) and have been related to changes in cell proliferation (Klezovitch et al., 2004; Machon et al., 2003). To determine whether loss of Pak4 causes abnormalities in the neuroepithelium, brain sections were stained with β-catenin antibody (Fig. 8). At E14.5, both in control and Pak4cKO brains, the β-catenin signal was highly concentrated at the ventricular interface, showing normal adherens junctions lining the lateral ventricles. At E18.5, however, β-catenin staining could no longer be seen at the ventricular interface of Pak4cKO brains, suggesting that Pak4 is required for normal neuroepithelial adherens junctions. Discussion Conventional Pak4 knockout mice die in utero by embryonic day E11.5. Examination of the Pak4 null embryos suggests that Pak4 has an important role in the development of the nervous system, although the most likely cause of death is a defect in the heart and extraembryonic tissue (Qu et al., 2003; Tian et al., 2009). To study the role for Pak4 in the nervous system in more depth and at later developmental stages, we generated conditional knockout mice in which Pak4 was deleted specifically in the nervous system. This was

accomplished by crossing mice with a floxed allele of Pak4, with transgenic mice that contain the Cre recombinase under the control of a nestin promoter (Tronche et al., 1999). We found that Nestin-Cre; Pak4f/Δ mice (referred to as Pak4cKO mice) were born alive but died prematurely, 2–4 weeks after birth. Abnormalities in the brains of Pak4cKO embryos and mice revealed that Pak4 is indispensable for normal development of a functional brain. Examination of brain sections and cultured cells indicated that Pak4 is essential for the development of the mammalian brain. In the developing brain, neurons derive from neuroepithelial cells within the ventricular zones. Neuroepithelial cells serve as neural progenitors, differentiating into either neurons or glial cells. Cortical neurogenesis occurs during embryonic development in animals. In contrast, glial cell generation occurs to a large extent after birth (Qian et al., 2000). Both cortical neurons and glia derive from the same cortical multipotent progenitor cells in the ventricular zone. As time progresses, some cells commit to a neural cell fate. Once neurogenesis starts, neuroepithelial cells undergo asymmetric cell division producing daughter cells with different cell fates. One daughter cell keeps the characters of neuroepithelial cells, while the other differentiates into a post-mitotic neuron or an intermediate progenitor, which produces two neurons after the next cell division in the subventricular zone (Haubensak et al., 2004; Miyata et al., 2004; Noctor et al., 2004). Newly generated neurons migrate towards the pial side, forming the neocortex with its lamination architecture (Gupta et al., 2002; Marin and Rubenstein, 2003). In the mouse cerebral cortex, neurogenesis starts at E11 and is largely finished around the time of birth. Some neurogenesis, however, can also continue into adulthood. In our study, impaired neurogenesis in the Pak4cKO mice was observed as early as embryonic day E16.5, although the differences in the brain structure became more dramatic starting around the time of birth. Consistent with a defect in neural progenitor cell proliferation, the

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Fig. 7. Mitosis at the ventricular surface was decreased in Pak4cKO brains. (A) Coronal sections of control and Pak4cKO brains at E18.5 were stained with the mitotic marker PH3 antibody (green) and DAPI (blue). Arrowheads indicate the dividing cells at the ventricular surface (VS) and arrows indicate dividing cells away from VS (nonVS). Cell division at the VS of the Pak4cKO brains was dramatically reduced, compared to control brains. Scale bar: 100 μm. (B) Quantitation of PH3-positive (PH3+) cells per field for E14.5–E18.5 brains. Starting at E16.5, significant decrease (*P b 0.05 or **P b 0.01) was found in the number of PH3+ cells in the Pak4cKO brains compared with the VS of the control brains. No significant difference (P N 0.05) was found in nonVS areas of control and mutant brains at these stages.

Pak4cKO mice exhibited a dramatic thinning of the cortex. The thickness of each layer of the cortex was reduced in the Pak4cKO mice compared to the controls, with the outer layers (layers II–IV), being affected most severely. Interestingly, however, although the cortical layers were abnormally thin in the Pak4cKO brains, none of the layers were lost. Each of the six cortical layers could be detected in the Pak4cKO brains. During the development to the nervous system there is initially a large amount of cell proliferation, as thousands of new neurons form. This leads to the formation of the vesicles that will form the forebrain, midbrain, and hindbrain. As development proceeds, the cells in each vesicle continue to multiply, and the specialized compartments within the brain develop. During this process the forebrain gives rise to the cerebrum. Cells in the cerebrum continue to proliferate until the structure reaches its full size, even after birth. This process of cell proliferation brings about an excess of both neurons and glia, and the later periods of brain development therefore also involve a large amount of cell death in which the excess cells undergo apoptosis. The final outcome is a brain of the correct size, where neurons that make their proper connections survive. In a number of gene knockout mice, defective proliferation or apoptosis has been related to abnormal development of the brain (Camarero et al., 2006; Cappello et al., 2006; Ke et al., 2007; Klezovitch et al., 2004; McFarland et al., 2006; Tomita et al., 2003; Zhang et al., 2006). Pak4 has been implicated in both cell proliferation and resistance to apoptosis (Gnesutta and Minden, 2003; Gnesutta et al., 2001; Li and Minden, 2005; Liu et al., 2008; Qu et al., 2001). We therefore reasoned that deletion of Pak4 may either inhibit cell proliferation or increase the amount of apoptotic cells. We did not see increased apoptosis at any stages in the Pak4cKO brains, however,

suggesting that cell survival was not affected by the lack of Pak4. In contrast, we observed a significant decrease in proliferation of neural progenitors starting before E16.5. Decreased proliferation level in the Pak4cKO brain was not due to changes in cell cycle rate or cell cycle withdrawal. Decreased proliferation of neural progenitors can result in impaired neurogenesis and can affect subsequent cortical lamination during later development. The mammalian cortex forms “inside-out” with the earliest born neurons occupying deeper layers in the brain and later born neurons in more superficial layers (Kubo and Nakajima, 2003). Decreased proliferation of the neural progenitors at later embryonic stages would therefore result in a reduction of later born neurons impacting the thickness of outer cortical layers more than earlier born neurons in deeper layers. This is consistent with what we observed in Pak4cKO brains, where decreased proliferation was not observed until approximately embryonic day E16.5, and the outer cortical layers were affected more severely than the inner layers. In addition to the decrease of proliferation in cortical progenitors, we also observed that proliferation in the subventricular zone facing the striatum was dramatically reduced in the Pak4cKO brains. The striatal subventricular zone has become a hot topic since it is a major region related to adult neurogenesis (Lim and Alvarez-Buylla, 1999; Lledo et al., 2008). This special zone contains a number of neural progenitors which continually produce new neurons throughout adulthood (see reviews: Altman and Das, 1967; Alvarez-Buylla and Garcia-Verdugo, 2002). These newly generated young neurons migrate to the olfactory bulb via the rostral migratory stream (Doetsch and Alvarez-Buylla, 1996; Kornack and Rakic, 2001; Lois and Alvarez-Buylla, 1994). A fraction of the cells that reach the olfactory bulb survive to complete their differentiation.

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Fig. 8. Loss of neuroepithelial adherens junctions in the Pak4 mutant brains. Coronal sections of brains were stained with β-catenin antibody to visualize adherens junctions (red) and DAPI (blue). In control brains, the staining of β-catenin was highly concentrated at the ventricular interface (indicated by arrows), showing neuroepithelial adherens junctions lining the lateral ventricle (LV). At E14.5, Pak4cKO brains showed normal neuroepithelial adherens junctions. By E18.5, however, β-catenin staining at the ventricular interface was hardly seen in the Pak4cKO brains (indicated by arrowheads). Scale bars: 100 μm.

This process is usually more conspicuous in the early postnatal brain (Luskin, 1993). In our study, the number of proliferating striatal progenitors was dramatically lower in the Pak4cKO mice by the time the mice were born, which could directly affect the neurogenesis after birth. Although the significance of the continual production of new neurons during postnatal life and the impact on the entire nervous system is still not clearly understood, we can speculate that the loss of proliferating progenitors in the striatal subventricular zone found in our Pak4cKO mice could in part contribute to the subsequent malformation of the brain and death of the mice. Decreased proliferation and neurogenesis in the Pak4cKO brains were associated with a thinning of all 6 layers of the cortex. As the mice developed, this corresponded to the onset of severe hydrocephalus, although hydrocephalus was evident only after birth, quite a bit later than the onset of the proliferation defect. Hydrocephalus can have many causes and it is not yet clear whether it is a result of the proliferation defects we observed, or whether it is the cause of death in the Pak4cKO mice. Hydrocephalus is often due to overproduction, poor absorption or blockage of cerebral spinal fluid (CSF). This can involve poor circulation and abnormalities in the vessels which carry the fluid. In some knockout mice that exhibit hydrocephalus, abnormal vessel formation has been detected (Tomita et al., 2003). The Pak4cKO mice did not show defects in vessel formation, however, either on the brain surface or around the ventricles up to P7, indicating that Pak4 is not required for the production of angiogenic signals from neural cells (not shown). Neuroepithelial cells in the ventricular zone develop apical tips at the ventricle side with processes extending from the cell body. These apical tips are connected at the ventricular interface by adherens junctions. In our study, the neuroepithelial adherens junctions, identified by β-catenin staining, could no longer be seen at the ventricular interfaces of Pak4 mutant brains at E18.5. Neuroepithelial adherens junctions have been proposed to be associated with cell proliferation (Klezovitch et al., 2004; Machon et al., 2003). In some types of stem cells, adherens junctions have also been reported to play a crucial role in the maintenance of proliferative status by maintaining the stem cell niche (Fuchs et al., 2004; Song et al., 2002; Yamashita

et al., 2003). Loss of adherens junctions could therefore be a factor contributing to the reduced cell proliferation level observed in Pak4 mutant brains. Our results show that Pak4 clearly has an important function in the development of the mammalian brain. An important question remaining is which signaling pathways mediate Pak4's functions in the brain. Pak4 has been implicated in a number of different signaling pathways in many different types of cells (Cammarano et al., 2005; Dan et al., 2001; Gnesutta et al., 2001; Li and Minden, 2005; Liu et al., 2008, 2010; Wong et al., 2010). We carried out Western blot analysis of Pak4 null and wild-type neurospheres, to determine whether some of these pathways are affected by the lack of Pak4 in these cells. Surprisingly, no significant change was found in any of the signaling proteins that we examined by Western blotting. These included phosphorylated forms of Erk, A-Raf, B-Raf, Raf-1, Stat3, c-Myc, p-120, as well as total LIMK1/2, and p120 (data not shown). These results indicate that at least in the nervous system, other signaling pathways may play important roles downstream to Pak4. Another possibility is that differences are in restricted parts of the brain, as we saw for nestin, PH3, and adherens junctions at the ventricular surface. The neuroepithelial cells located at the ventricular surface are highly polarized and undergo interkinetic nuclear migration (INM) and asymmetric cell division. Adherens junctions have important roles in the neuroepithelium, in establishing and maintaining cell polarity and in maintaining cell shape by organizing the actin cytoskeleton. Adherens junctions are composed of proteins such as catenins and cadherins. We found that the β-catenin signal for adherens junctions at the ventricular interface was lost in Pak4 mutant brains. It has been reported that disruption of β-catenin in the brain also causes hydrocephalus and postnatal death (Ohtoshi, 2008), as well as decreased cell proliferation (Machon et al., 2003). This opens up the possibility that Pak4 may be involved in the β-catenin signaling pathway, thereby contributing to the maintenance of neuroepithelial adherens junctions. Future identification of new Pak4 substrates and downstream signaling pathways will provide important new information about how Pak4 affects adherens junction formation, neuronal cell proliferation, and brain development.

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In summary, we have found that loss of Pak4 in the nervous system causes inhibition of cell proliferation and neuro-differentiation, eventually leading to a thinning of all 6 layers of the cortex. Our work establishes that Pak4 has a critical role in the proliferation of neural progenitors. This is consistent with previous work showing that Pak4 has key roles in cell proliferation and survival (Gnesutta and Minden, 2003; Gnesutta et al., 2001; Li and Minden, 2005; Liu et al., 2008; Qu et al., 2001) and provides important new information about the role for Pak4 in the developing nervous system.

Acknowledgments This work was supported by NIH grant R01 MH065265-01 and a grant from the New Jersey Spinal Cord Commission, to AM. We thank Renping Zhou for valuable suggestions on this project.

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