Discs large 5 is required for polarization of citron kinase in mitotic ...

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Discs large 5 is required for polarization of citron kinase in mitotic neural precursors YoonJeung Chang,1 Olga Klezovitch,2 Randall S. Walikonis,1 Valera Vasioukhin2 and Joseph J. LoTurco1,* Department of Physiology and Neurobiology; University of Connecticut; Storrs, CT USA; 2Division of Human Biology; Fred Hutchinson Cancer Research Center; Seattle, WA USA

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Key words: neocortex, citron kinase, discs large 5, neurogenesis, ventricular zone, cell polarity Abbreviations: CitK, citron kinase; Dlg5, discs large 5; VZ, ventricular zone; PDZ, PSD95-discs large-ZO-1; MAGUK, membrane-associated guanylate kinase

Citron kinase (CitK), a protein essential to neurogenic cell division in the central nervous system, is highly polarized in neural progenitors. The mechanisms that polarize CitK to cellular domains that line the ventricular surface of neuroepithelium are currently not known. Here we report that Discs large 5 (Dlg5), a member of the MAGUK family, is an interactor of CitK required for CitK polarization. The CitK-Dlg5 interaction was first revealed in a protein array screen of proteins containing PDZ domains, and then subsequently confirmed by co-immunoprecipitation. Moreover, in Dlg5-/mice CitK fails to polarize in mitotic neuronal precursors. In addition, the total number of mitotic progenitors and the ratio of ventricular to abventricular mitotic progenitors in developing neocortex are significantly decreased in Dlg5-/- embryos. Dlg5 is therefore required to maintain the polarization of a protein essential to neurogenic cytokinesis, and plays a role in localizing cell divisions to the surface of the lateral ventricles in embryonic brain.

Introduction Neurogenic cell divisions in the developing neocortex are located either along the surface of the ventricular zone, ventricular divisions, or away from the ventricular surface, abventricular divisions. During the period of neuronal production ventricular cell divisions are primarily stem-like divisions that give rise to a fated neuronal precursor that migrates away from the ventricular surface and a radial progenitor that remains attached to the ventricular surface.1,2 The abventricular cell divisions, in contrast, are thought to primarily give rise to two postmitotic neuronal precursors.1-3 The final number of neurons generated in the neocortex is largely determined by changes in the numbers of these two types of cell divisions over time. Mutations in citron kinase (CitK) in rodents revealed that CitK is essential to neurogenic cell divisions in developing central nervous system. Both mice and rats with null mutations of CitK are microcephalic, and display binucleated cells, cytokinesis failure, and massive apoptosis in proliferative zones of embryonic neocortex.4,5 The entire molecular pathway of CitK in the control of neural progenitor cell division remains incomplete. RhoA is known to bind to CitK, enhances CitK kinase activity, and this activation is required for translocation of CitK from midzone spindles to cleavage furrows.6,7 At the end of cytokinesis CitK becomes highly concentrated at the midbody ring, where it colocalizes with the human microcephaly protein ASPM.8 To date,

the only identified substrate of CitK, although not yet confirmed in vivo, is the regulatory light chain of myosin II.9 Citron kinase protein is highly polarized in dividing neural progenitor cells at the ventricular surface in the embryonic nervous system. The protein is localized to end feet, cleavage furrows and midbody rings that line the ventricular surface of the lateral ventricles.4 The mechanisms that polarize CitK to the ventricular surface are not known, however the presence of a consensus QSSV PDZ binding domain at the C-terminus of CitK raised the possibility that it may interact with one of the polarity genes that contain PDZ domains. Mammalian Discs large 5 (Dlg5) is a membrane-associated guanylate kinase (MAGUK) protein containing four PDZ domains.10 MAGUK proteins are known to regulate cellular adhesion and plasticity at the developing junctional complex, and Dlg1 is part of the polarity protein complex at the baso-lateral membrane of mammalian cells.11 A recent study has shown that Dlg5 binds to β-Catenin at sites of cell-cell contact and may act as a scaffolding protein.12 Importantly, Dlg5 is highly expressed in developing brain, and is required for the maintenance of adherens junctions and the maintenance of cell polarity in the developing mammalian brain and kidney.13 In this study, we report that Dlg5 interacts with CitK, and that Dlg5 plays a role in the polarization of CitK and the location of mitotic neural progenitors. The C-terminus of CitK can directly bind to two PDZ domains within Dlg5, and Dlg5

*Correspondence to: Joseph J. LoTurco; Email: [email protected] Submitted: 11/02/09; Revised: 01/22/10; Accepted: 03/08/10 Previously published online: www.landesbioscience.com/journals/cc/article/11730 1990

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co-immunoprecipitates with CitK in embryonic neocortex. CitK fails to polarize to the ventricular zone (VZ) surface of mitotic neural progenitors in Dlg5-/- mutants. In addition, Dlg5-/- neuroepithelium shows a reduction in the number of ventricular cell divisions, and an increase in abventricular cell divisions. Taken together, we hypothesize that Dlg5 interacts with CitK during neurogenesis to maintain ventricular positioned cell divisions. Results Citron kinase interacts with discs large 5. Citron kinase (CitK) contains a consensus PDZ binding motif at its C-terminus, and it has not been previously determined with what specific PDZ domains CitK binds. To identify such direct interactions we used the PanomicsTM PDZ domain array to screen for specific binding between CitK, and characterized the binding to PDZ domains contained in 23 different proteins. The PDZ domain array contains 33 GST-tagged PDZ domains from 23 proteins. For this screen we used a purified HIS-tagged C-terminal peptide of CitK (324 a.a.) as a probe, and identified binding of this peptide probe to the array with an antibody against the HIS epitope. We found that the affinity purified C-terminal peptide of CitK bound to only three peptides on the array (Fig. 1A). The two strongest signals were for PDZ domains 2 and 3 of Dlg5 (Dlg5-D2 and Dlg5-D3) (first panel in Fig. 1A) and a weaker signal was apparent for a PDZ domain in LOMP (LMO7 LIM domain 7) (fifth panel in Fig. 1A). The CitK peptide failed to bind to any other of the other 30 PDZ domains on the array including PDZ domains 1 and 4 of Dlg5 (second panel in Fig. 1A) and PDZ domains of Dlg3 (third panel in Fig. 1A). These results indicate that the C-terminus of CitK can bind to two different PDZ domains of Dlg5 with relatively high specificity. This screen also identifies the first direct interaction between CitK and PDZ domains contained within any protein. The array results indicated a potential direct interaction between CitK and Dlg5, and we therefore performed co-immunoprecipitation and immunocytochemistry experiments to determine whether an interaction was present in neural progenitor cells. To test for interaction between endogenous Dlg5 and CitK in developing forebrain, we performed co-immunoprecipitation experiments of Dlg5 and CitK from E12, E15 and E18 embryonic forebrain (Fig. 1B). In lysates from E12 brain, an anti-Dlg5 antibody13 immunoprecipitated Dlg5 (second panel in Fig. 1B) and co-immunoprecipitated CitK (first panel in Fig. 1B). The amount of CitK that co-immunoprecipitated with the anti-Dlg5 antibody in E15 and E18 brain homogenates was decreased relative to that in E12. This decrease in CitK co-immunoprecipitate was correlated with a general decrease in the amount of CitK in the input lysate (third panel in Fig. 1B). As a test for specificity for the co-immunoprecipitation, we found that CitK did not co-immunoprecipitate with either a rabbit IgG (fourth panel in Fig. 1B) or a rabbit anti-BLBP antibody (fifth panel in Fig. 1B). In addition, as a positive control for Dlg5 co-immunoprecipitation, we found that the anti-Dlg5 antibody co-immunoprecipitated b-Catenin in developing brain (sixth panel in Fig. 1B) as previously shown.12,13 To further confirm specificity in the co-ip experiments we found

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that another protein that has been associated with cytokinesis in neural progenitors and localizes to cytokinesis furrows and midbodies, AuroraB, did not co-immunoprecipitate with Dlg5 (last panel in Fig. 1B). Additional support for an in vivo interaction between Dlg5 and CitK in neural progenitors came from colocalization in double label immunostaining in cultured neural progenitor cells (Fig. 1C). In isolated neural progenitor cultures the co-localization of immunopositivity for CitK and Dlg5 was largely restricted to the midbody ring, the site of strongest CitK positivity in isolated cell cultures. The co-immunoprecipitation and immunocytochemistry results suggest that endogenous Dlg5 and CitK interact within neural progenitors in vivo and in cell culture. Dlg5 is required for polarization of CitK to the VZ surface. In order to test for a functional requirement of Dlg5 in CitK localization we assayed CitK protein levels and distribution in Dlg5 mutants and controls. The Dlg5 knockout mice (Dlg5-/-) used were previously generated and characterized in a paper which showed a disruption in polarization of cells in the third ventricle of developing brain.13 We used these same mutant mice to determine whether CitK expression is altered by Dlg5 loss in the lateral ventricles. First we tested whether CitK expression levels were altered in the mutants. Immunoblots of protein lysates from the forebrains of E15.5 wildtype (Dlg5 +/+), Dlg5 homozygous mutant (Dlg5-/-), and heterozygous mutants (Dlg5 +/-) were probed with an anti-CitK antibody.14 We found that the total levels of CitK protein were not altered in Dlg5-/- forebrains at E15.5 in homozygous mutants relative to heterozygous or wildtype littermates (first panel in Fig. 1D). Next, we analyzed E15.5 Dlg5-/- brains by immunohistochemistry and confocal analysis for alterations in CitK distribution. CitK immunostaining showed more diffuse localization at the VZ surface in the Dlg5-/- neocortex (green, Fig. 2A, lower) compared to wildtype neocortex (green, Fig. 2A, upper). Moreover, the number of CitK puncta (arrows) that typically decorate the VZ surface was greatly reduced in Dlg5-/- neocortex (green, Fig. 2A, lower). Although we found that the VZ surface localization of CitK was nearly completely disrupted in the Dlg5 mutant neocortex, we observed that adherens junction markers normally polarized to the VZ surface remained intact. Specifically, immunocytochemistry for ZO-1 (Fig. 2A), PKCl, and b-Catenin (Fig. 2B) remained highly polarized along the VZ surface in Dlg5-/- brains in a pattern similar to that in wildtype brains (Dlg5 +/+). Thus, while CitK polarization is disrupted in the Dlg5 mutant, this does not appear to be a consequence of a general disruption in cellular polarity in neural progenitors in the lateral ventricle. Dlg5 is required for CitK polarization during metaphase. In previous studies, polarized CitK puncta were found to be at midbody rings, cytokinesis furrows and endfeet of neural progenitors at the ventricular surface in vivo.4,8 In Dlg5-/- mice, although far fewer in number, CitK immunopositve puncta were distributed along the VZ surface. These puncta tended to be larger in size than the majority of puncta in wildtype brain and also appeared to have morphologies similar to the midbody ring at the center of the cytokinetic midbody. In order to determine whether

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Figure 1. CitK interacts with Dlg5. (A) Interaction between the PDZ-binding domain of CitK and the PDZ domain-containing proteins. A PDZ domain array containing PDZ domains from 23 proteins was probed with a purified recombinant C-terminal peptide of CitK. The CitK probe bound to PDZ domain 2 and 3 of Dlg5 (Dlg5-D2 and Dlg5-D3, top), while it did not bind to PDZ domain 1 and 4 of Dlg5 (Dlg5-D1 and Dlg5-D4, second). HIS, immobilized His-tag only served as a positive control; PIST, GOPC (NP_065132); GEF2, RAPGEF6 (NP_057424); LIK1, LIMK1 (NP_002305); GEF11, ARHGEF11 (NP_055599); GEF12, ARHGEF12 (NP_056128). (B) Interaction between endogenous CitK and Dlg5 in rat developing brain. A rabbit polyclonal anti-Dlg5 antibody was used for immunoprecipitation and a mouse anti-CitK antibody (CRIK) was used for western blotting to detect the interactions. In rat embryonic brain lysates (E12, E15, E18), an anti-Dlg5 antibody immunoprecipitated Dlg5 (second) and CitK co-immunoprecipitated with Dlg5 (first). Note that coimmunoprecipitated CitK was gradually decreased in E15 and E18 brain due to decreased amount of endogenous CitK (third). CitK did not co-immunoprecipitate with a rabbit IgG (fourth) and a rabbit polyclonal BLBP antibody (fifth). b-Catenin co-immunoprecipitated with Dlg5 (sixth), while AuroraB did not (seventh). (C) During cytokinesis of cortical progenitor cells in dissociated cell culture, Dlg5 (green) and CitK (red) immunopositivity co-localize to the midbody ring. A confocal orthogonal projection shows the overlap of the CitK and Dlg5 immunopositive signals at the midbody ring of neural progenitors. (D) CitK was expressed in E15.5 Dlg5-/- mice brain as well as in Dlg5+/+ and Dlg5+/- brains (first). An anti-Dlg5 antibody confirmed that Dlg5 is absent in E15.5 Dlg5-/- mice brain (second). a-tubulin was used as internal control (third).

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Figure 2. Dlg5 deficient neocortex displays less CitK polarized to the VZ surface. (A) The E15 wildtype littermate (Dlg5+/+, upper) neocortex shows highly polarized CitK (green, arrows) at the VZ, while the E15 Dlg5-/- mice neuroepithelium (Dlg5-/-, lower) shows that CitK (green, arrows) is diffuse throughout the cell and is less aggregated at the VZ surface. ZO-1 (red) labels the ventricular zone surface. Scale bars, 10 µm. (B) PKCl and b-Catenin immunostaining of the E15 Dlg5-/- neocortex (lower) shows that the polarity of the adherens junction is maintained in the lateral ventricles in the absence of Dlg5. Scale bars, 50 µm. (C) In the Dlg5-/- mice the remaining CitK immunopositivity at the VZ surface of the lateral ventricles is localized to the midbody ring between progenitor cells in late cytokinesis. Scale bar, 10 µm.

indeed these remaining polarized CitK immunopositivities were between dividing cells, we double-labeled with phospho-vimentin55 (phVim) and CitK antibodies. Phospho-vimentin immunopositivity reliably labels pairs of dividing cells at the VZ surface. We found that phVim (+) cell pairs, which correspond to cells in late telophase, reliably had a CitK positive puncta in the position between the two cells in Dlg5-/- mutants as well as controls


(arrow in Fig. 2C). This result indicates that Dlg5 is not required for localization of CitK to the midbody ring in vivo. Considering the maintained localization of CitK to the midbody ring, and yet overall decrease in polarized CitK to the VZ surface, we next assessed whether CitK polarization was maintained just prior to cytokinesis. Metaphase cells were identified by a combination of phVim (red) immunostaining that labels M-phase cells and TO-PRO®-3 iodide (TO-PRO) to label the condensed chromosomes of cells in metaphase. Triple staining for CitK, phVim and TO-PRO revealed that CitK (green) was polarized to the VZ surface in metaphase cells of wildtype as recently reported.15 In contrast, there was a complete lack of VZ polarized CitK in metaphase cells in Dlg5-/- mutants (arrowheads) (Fig. 3A). We quantified the polarity of CitK immunopositivity as described in the methods and Figure 3B. The ratio of CitK expression at the VZ surface in the Dlg5-/- metaphase cells was 7-fold decreased compared to that in wildtype cells (n = 3 brains, 15 cells from each condition, Fig. 3C). Together, these results indicate that while Dlg5 function is not critical to the aggregation of CitK to the midbody ring, but is essential to CitK polarization at the VZ surface in metaphase. Absence of Dlg5 causes fewer M-phase cells at the VZ surface. In the developing neocortex there are both ventricular and abventricular mitoses that are believed to differentially produce symmetric and asymmetric cell divisions. We assessed the number of ventricular and abventricular M-phase cells by quantifying the number of phVim (+) cells in both wildtype and Dlg5-/- neocortex. The neuroepithelia of Dlg5-/- mice displayed a decreased number of ventricular mitoses (arrowheads), while the number of abventricular mitoses (arrows) was increased, as compared to wildtype neocortices (n = 3 brains, 5 sections from each brain, Fig. 4A and B). The proportion of abventricular divisions to ventricular divisions was significantly increased in Dlg5-/- brain (n = 3 brains, 5 sections from each brain, Fig. 4C). These results indicate that the absence of Dlg5 decreases the number of M-phase cells at the VZ surface. Discussion

In this study, we show that Dlg5 interacts with CitK, and that the C-terminus of CitK binds directly to PDZ domains 2 and 3 of Dlg5. Analysis of Dlg5 knockout mice further revealed a potential functional significance for this interaction. Specifically, polarization of CitK to the VZ surface is disrupted in Dlg5 knockouts, and the number of ventricular M-phase cells was decreased and non-surface mitoses increased in Dlg5-/- mutants. Previous studies have shown that MAGUKs are involved in the maintenance of epithelial polarity in Drosophila16 and in mammalian cells.17 The present data show that Dlg5 contributes to CitK polarity during the cell cycle of endogenous neural progenitors in mammalian neocortex

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Figure 3. Failure of CitK polarization in Dlg5-/- metaphase cells. (A) CitK (green) highly polarizes to the ventricular pole in cells in metaphase (arrowheads in top panels) in wildtype littermates (Dlg5+/+), while CitK is diffuse in metaphase cells in Dlg5-/- mutants. Scale bar, 10 µm. Metaphase cells are outlined, and ‘A’ indicates ventricular pole and ‘B’ indicates the abventricular pole of mitotic cells. (B) The schematic depicts the ventricular expression and the abventricular expression of CitK. The dotted lines represent the ventricular-abventricular axis between the ventricular pole (A) and the abventricular pole (B) and the perpendicular half line. (C) Dlg5-/- metaphase cells display nearly equal amount of CitK expression in ventricular and abventricular halves of the cells, while Dlg5+/+ metaphase cells maintain higher CitK expression in ventricular side (n = 3 brains for either wildtype or Dlg5-/-, 5 cells from each brain and total 15 cells were quantified for each condition). **p < 0.01, determined by Student’s t-test. Error bars represent ± s.e.m.

(Fig. 3), while CitK localization to the midbody ring is independent of Dlg5 (Fig. 2C). Nechiporuk et al.13 showed that loss of Dlg5 caused severe disruption in the polarization of three junctional proteins, ZO-1, β-Catenin and N-Cadherin, in cells lining the third ventricle of developing brain, but, similar to our results here, not to polarization of these markers in cells of the lateral ventricles. The loss of cellular polarization observed in this previous study in the third ventricle resulted in a fusion of the central aqueduct which caused subsequent hydrocephalus. We found in this study that loss of Dlg5 in the ventricular zone surrounding the lateral ventricles of developing neocortex did not disrupt polarization of ZO-1, PKCl or b-Catenin (Fig. 2A and B), but caused a more subtle loss in polarization of CitK. This apparent difference between third and lateral ventricular cells may indicate that Dlg5 has different levels of function in regulating polarity in different neural progenitor types, and shows that a more limited loss of polarity does occur with cells in the lateral ventricles as a result of Dlg5 loss of function. We found a significant change in the ratio of ventricular to abventricular cell divisions in the developing neocortex in Dlg5-/mice. This suggests that as a result of non-polarized CitK in metaphase that there may be a decrease in the number of ventricular

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stem-like divisions and a corresponding increase in abventricular, neuron-neuron cell divisions. Such a change could result in a premature depletion of progenitors and a corresponding decrease in the total number of neurons produced. The neocortex of Dlg5-/- mice is smaller and thinner after birth, suggesting a possible decrease in neural cell production consistent with this model, however, because there is also hydrocephaly as a result of the fusion of the third ventricle, and such hydrocephaly can cause secondary changes in cell number, it is not yet possible to conclusively determine that an apparent loss of cell number and volume in neocortex is the direct result of altered neurogenesis. A future conditional deletion of Dlg5 just in the forebrain may help to distinguish between these possibilities. Nevertheless, the present results reveal that polarity of a protein critical to neurogenic cell division, CitK, is dependent upon the presence of Dlg5. These results are also the first to our knowledge to show a direct interaction between a polarity control protein, Dlg5, and a cytokinesis control protein, CitK. Such a connection would be predicted to exist in neuroepithelia, and other epithelia, in which mitoses are frequently localized to the surface of epithelia. Dlg5 and CitK interaction may thus indicate presence of a larger signaling complex that regulates the positioning of mitoses at luminal surfaces of epithelia during development.

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Figure 4. Absence of Dlg5 decreases ventricular surface cell divisions. (A) Comparison of the E15.5 neocortex of Dlg5+/+ and Dlg5-/- mice. Fewer Mphase cells (arrowheads) are found at the ventricular surface in E15 Dlg5-/- neuroepithelium (right) compared to wildtype littermates (Dlg5+/+, left). In addition, more non-surface M-phase cells (arrows) are present in the abventricular zone in Dlg5-/- neuroepithelium than in Dlg5+/+. Scale bar, 50 µm. (B) Quantification shows the comparison between the number of M-phase cells at the ventricular surface and abventricular region in Dlg5-/- and Dlg5+/+ embryonic neocortex. The phVim (+) cells were counted in a 200 mm wide radial traverse through the dorsal cortex (n = 3 brains, 5 sections from each brain). (C) Graph shows that the percentage of non-surface M-phase cells is significantly increased in Dlg5-/- neocortex (n = 3 brains, 5 sections from each brain). **p < 0.01; *p < 0.1, determined by Student’s t-test. Error bars represent ± s.e.m.

Materials and Methods Protein domain array system. The PDZ domain array IV from PanomicsTM was used to screen proteins interacting with the C-terminus of CitK which includes a terminal QSSV consensus PDZ binding domain. The array membranes were blocked by


incubation in 5% nonfat dry milk in TBST for 2 hrs. Sequence coding for the C-terminal 324 residues of CitK (amino acids 1732–2056) were cloned into the vector pET32a (Novagen, Germany). The resulting HIS tagged protein was expressed in Escherichia coli DH5a and affinity purified on Ni-NTA His•Bind Resin. Purified HIS-tagged CitK (pET32a-HIS-CitK-

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PDZ/SH3) was diluted in TBST to a concentration of 10 mg/ ml, and incubated with the array membrane overnight at 4°C. The membrane was then washed three times in TBST and subsequently incubated with a rabbit polyclonal anti-HIS antibody for 3 hrs. After washing three times with TBST, the membrane was incubated with a goat anti-rabbit antibody, and washed three times with TBST and three times with TBS. Bound antibodies were detected with ECLTM (GE Healthcare, Piscataway, NJ). PDZ domains from the following 23 proteins were included on the array: MPDZ (NP_003820), DLG2 (NP_001355), DLG3 (NP_066943), DLG5 (NP_004738), PARD6B (BAB40756), LIMK1 (NP_002305), LMO7 (NP_005349), PDLIM4 (NP_003678), PDLIM3 (NP_055291), TIAM1 (NP_003244), LIN7C (NP_060832), LIN7B (NP_071448), LIN7A (NP_004655), ARHGEF11 (NP_055599), ARHGEF12 (NP_056128), PDZK1 (NP_002605), SNTB1 (NP_066301), SNTA1 (NP_003089), SHANK1 (NP_057232), MPP6 (NP_057531), GOPC (NP_065132), RAPGEF6 (NP_057424) and RIMS2 (NP_055492). Co-immunoprecipitation of Dlg5 and CitK. Lysates from rat forebrain at embryonic day 12 (E12), embryonic day 15 (E15) and embryonic day 18 (E18) were subjected to IP with a rabbit polyclonal anti-Dlg5 antibody13 followed by immunoblotting with a mouse monoclonal anti-CitK antibody (1:1,000, BD Pharmingen), a rabbit polyclonal b-Catenin antibody (1:10,000, Sigma), and a mouse anti-AuroraB antibody (1:250, BD Pharmingen) for detecting interactions and a rabbit polyclonal anti-Dlg5 and a mouse monoclonal anti-CitK antibody for detecting inputs. Rabbit IgG (Sigma) and a rabbit polyclonal BLBP antibody (Abcam) were used for control immunoprecipitation. Animals. Dlg5 knockout (Dlg5-/-) mice were generated as previously described.13 Heterozygous (Dlg5 +/-) pairs of the Dlg5 knockout mice were time-mated and E15.5 mouse brains were harvested and prepared for cryosection. The genotyping was done as described in Nechiporuk et al.13 All animal care procedures were approved by the University of Connecticut IACUC (the protocol number A06-026). Immunostaining on cultured cells. Primary cultures of progenitor cells from E11 rat neocortex were grown to 50% confluency in a serum-free medium consisting of DMEM with L-glutamine, sodium pyruvate, B-27, N-2, bFGF2 and EGF (all from Invitrogen). After a brief fixation with cold methanol, cells were subjected for standard immunostaining. The following antibodies were used: a rabbit polyclonal anti-Dlg5 antibody13 (1:100), a mouse monoclonal anti-CitK antibody (1:200, BD Pharmingen), a goat anti-rabbit IgG conjugated Alexa 488 (Invitrogen), and a goat anti-mouse IgG conjugated Alexa 568 (Invitrogen). Immunohistochemistry. Embryonic day 15.5 mice brains were harvested in cold PBS and fixed with 0.5% paraformaldehyde in References 1.

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Pontious A, Kowalczyk T, Englund C, Hevner RF. Role of intermediate progenitor cells in cerebral cortex development. Dev Neurosci 2008; 30:24-32. Noctor SC, Martínez-Cerdeño V, Kriegstein AR. Contribution of intermediate progenitor cells to cortical histogenesis. Arch Neurol 2007; 64:639-42.

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PBS, cryoprotected, and sectioned on a cryostat at 10–20 µm in the coronal plane. Sections were permeablized/blocked in 1X PBS with 0.5% TritonX-100 and 5% normal goat serum for 1 hr at room temperature, and immunostained with primary antibody solution (0.2% TritonX-100, 2.5% NGS in 1X PBS) overnight at 4°C. The following primary antibodies were used: a rabbit polyclonal anti-Dlg5 antibody13 (1:100), a mouse monoclonal anti-CitK antibody (1:200, BD Pharmingen), a rabbit polyclonal anti-CitK antibody (CT295,14 1:200), a rat monoclonal anti-ZO-1 antibody (1:300, Development studies Hybridoma bank at the University of Iowa), a mouse monoclonal anti-PKCl antibody (1:100, BD Pharmingen), a rabbit polyclonal anti-bCatenin (1:500, Sigma), and a mouse monoclonal anti-phosphovimentin55 (1:500, MBL). After three washes (10 min each) with the rinse solution (0.2% TritonX-100, 2.5% NGS in 1X PBS), the sections were incubated with secondary antibodies (0.2% TritonX-100, 2.5% NGS in 1X PBS) for 2 hrs at room temperature. The following secondary antibodies were used: a goat anti-rabbit IgG conjugated Alexa 488 (1:200, Invitrogen), a goat anti-mouse IgG conjugated Alexa 568 (1:200, Invitrogen), a goat anti-rat IgG conjugated Alexa 568 (1:200, Invitrogen), a goat anti-mouse IgG conjugated Alexa 488 (1:200, Invitrogen), and a goat anti-rabbit IgG conjugated Alexa 568 (1:200, Invitrogen). Then the sections were washed with the rinse solution 5 times, and DAPI (1:3,000, Sigma) and TO-PRO®-3 iodide (1:3,000, Molecular probes) were added in the third wash for counter staining. The sections were mounted with Prolong antifade (Invitrogen). Imaging and statistical analysis. All double- or triple-labeling was viewed with a laser-scanning confocal microscope (Leica TCS SP2, laser lines at 488 nm, 543 nm, 633 nm) using 20X PL APO N.A. 0.7, 40X PL APO N.A. 1.25 and 100X PL APO N.A. 1.40 oil immersion objectives. We used Adobe Photoshop CS3 to assemble all images. CitK immunopositivity (Fig. 3) was quantified in ImageJ. First, the metaphase cells were outlined, ventricular and abventricular poles were assigned based on metaphase plate indicated by TO-PRO®-3 iodide staining. The half line was operationally defined as the middle of the ventricularabventricular axis. Then, CitK staining was measured in ventricular or abventricular side with the histogram tool and the ratio was calculated as ventricular expression/abventricular expression. We used two-sample Student’s t-test to compare means of two independent groups and ANOVA. We considered values as significant when p < 0.05. All data are presented as means. Error bars represent ± s.e.m. Acknowledgements

We thank Dr. Mary Kennedy and Leslie Schenker for the anticitron antibody (CT295). This work has been supported by NIH grant MH056524 to J.J.L. and CA131047 to V.V.

Götz M, Huttner WB. The cell biology of neurogenesis. Nat Rev Mol Cell Biol 2005; 6:777-88. Sarkisian MR, Li W, Di Cunto F, D’Mello SR, LoTurco JJ. Citron-kinase, a protein essential to cytokinesis in neuronal progenitors, is deleted in the flathead mutant rat. J Neurosci 2002; 22:217.

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Di Cunto F, Imarisio S, Hirsch E, Broccoli V, Bulfone A, Migheli A, et al. Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis. Neuron 2000; 28:115-27. Eda M, Yonemura S, Kato T, Watanabe N, Ishizaki T, Madaule P, Narumiya S. Rho-dependent transfer of Citron-kinase to the cleavage furrow of dividing cells. J Cell Sci 2001; 114:3273-84.

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Di Cunto F, Calautti E, Hsiao J, Ong L, Topley G, Turco E, Dotto GP. Citron rho-interacting kinase, a novel tissue-specific ser/thr kinase encompassing the Rho-Rac-binding protein Citron. J Biol Chem 1998; 273:29706-11. 8. Paramasivam M, Chang YJ, LoTurco JJ. ASPM and citron kinase co-localize to the midbody ring during cytokinesis. Cell Cycle 2007; 6:1605-12. 9. Yamashiro S, Totsukawa G, Yamakita Y, Sasaki Y, Madaule P, Ishizaki T, et al. Citron kinase, a Rhodependent kinase, induces di-phosphorylation of regulatory light chain of myosin II. Mol Biol Cell 2003; 14:1745-56. 10. Anderson JM. Cell signalling: MAGUK magic. Curr Biol 1996; 6:382-4.


11. Funke L, Dakoji S, Bredt DS. Membrane-associated guanylate kinases regulate adhesion and plasticity at cell junctions. Annu Rev Biochem 2005; 74:219-45. 12. Wakabayashi M, Ito T, Mitsushima M, Aizawa S, Ueda K, Amachi T, Kioka N. Interaction of lp-dlg/ KIAA0583, a membrane-associated guanylate kinase family protein, with vinexin and β-catenin at sites of cell-cell contact. J Biol Chem 2003; 278:21709-14. 13. Nechiporuk T, Fernandez TE, Vasioukhin V. Failure of epithelial tube maintenance causes hydrocephalus and renal cysts in Dlg5-/- mice. Deve Cell 2007; 13:338-50. 14. Zhang W, Vazquez L, Apperson M, Kennedy MB. Citron binds to PSD-95 at glutamatergic synapses on inhibitory neurons in the hippocampus. J Neurosci 1999; 19:96-108.

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15. Chang Y, Paramasivam M, Girgenti MJ, Walikonis RS, Bianchi E, LoTurco JJ. RanBPM regulates the progression of neuronal precursors through M-phase at the surface of the neocortical ventricular zone. Dev Neurobiol 2010; 70:1-15. 16. Siegrist SE, Doe CQ. Microtubule-induced Pins/ Galphai cortical polarity in Drosophila neuroblasts. Cell 2005; 123:1323-35. 17. Margolis B, Borg JP. Apicobasal polarity complexes. J Cell Sci 2005; 118:5157-9.

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