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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Development. Author manuscript; available in PMC 2011 January 5.
Published in final edited form as:
PMCID: PMC3016226
NIHMSID: NIHMS111454

MALS-3 regulates polarity and early neurogenesis in the developing cerebral cortex

Abstract

Apico-basal polarity plays an important role in regulating asymmetric cell divisions by neural progenitor cells (NPCs) in invertebrates, but the role of polarity in mammalian progenitor cells remains poorly understood. Here we characterize the function of the PDZ domain protein MALS-3 in the development of the cerebral cortex. We find that MALS-3 is localized to the apical domain of NPCs, where it is closely associated with the cell membrane. Analysis of mice lacking all three MALS genes revealed that MALS-3 is required for the normal apical localization of the polarity proteins PATJ and PALS1 but not for the formation or maintenance of adherens junctions. Loss of MALS altered early neurogenesis as evidenced by a decrease in BrdU incorporation at E11, which reflected a slower progression through the cell cycle. In addition, early-generated daughters of MALSTKO mutant NPCs showed an increase in cell cycle exit and differentiation into neurons. Interestingly, these effects were transient, since cycling progenitors recovered normal cell cycle properties a few days later. Gain-of-function experiments in which MALS-3 was targeted to the entire membrane resulted in a breakdown of both apico-basal polarity and adherens junctions, resulting in a disruption of the ventricular surface and the appearance of NPCs in the lateral ventricles. These results suggest that the maintenance of apico-basal polarity is required for normal neurogenesis.

Introduction

Neural progenitor cells (NPCs) in the cortical ventricular zone (VZ) are inherently polarized, with distinct cellular structures (cilia, centrosomes and adherens junctions) and molecular factors (Numb) localized apically (Aaku-Saraste et al., 1996; Astrom and Webster, 1991; Chenn et al., 1998; Zhong et al., 1996). This intrinsic polarity is exploited when a NPC undergoes asymmetric mitotic divisions to produce apical and basal daughters with different cell fates (Chenn and McConnell, 1995; Chenn et al., 1998; Gotz and Huttner, 2005; Huttner and Kosodo, 2005). Imaging studies have suggested that the apical cell surface is distributed unequally during asymmetric cell division of NPCs, suggesting that the molecular components localized apically may play a critical role in determining the fates and mitotic behaviors of newly generated daughter cells (Huttner and Kosodo, 2005; Kosodo et al., 2004).

The fate of the daughter cell that retains the apical process remains somewhat controversial. One set of studies suggest that this daughter cell continues to proliferate and remains a progenitor cell (Miyata et al., 2001), while other studies suggest that it differentiates into a neuron (Huttner and Kosodo, 2005; Kosodo et al., 2004; Wodarz and Huttner, 2003). Despite this disagreement, it is clear that only one of the two daughters of an asymmetric division inherits apical cell components, and that this daughter cell has a fate distinct from the other (Miyata et al., 2001; Noctor et al., 2001). These observations suggest that determinants found at the apical membrane (such as Par6 and Par3) or associated with adherens junctions may influence the fates of daughter cells (Cappello et al., 2006; Imai et al., 2006),.

In fact, several proteins such as Numb, PAR-3, Cdc-42, Prominin-1 (CD133), Aspm, and afadin that are localized apically in NPCs during cortical development (Gotz and Huttner, 2005) appear to play a critical role in corticogenesis via establishment of apico-basal polarity (Fish et al., 2006; Junghans et al., 2005; Petersen et al., 2002). For example, the conditional disruption of Cdc-42 causes not only a loss of other apical markers at the lumenal surface of NPCs, but also alters the positioning of dividing NPCs from an apical to a more basal region of the VZ due to a loss of adherens junctions, which alters the cell fate of those progenitors (Cappello et al., 2006). Transgenic mice that overexpress β-catenin, an apically localized protein, during corticogenesis displayed larger brains and the presence of gyri and sulci (normally absent in rodent brains) due to an increase in the number of cycling progenitors (Chenn and Walsh, 2003). Loss of the basally localized protein Lgl results in severe brain dysplasia and proliferation defects during corticogenesis (Klezovitch et al., 2004). These studies provide strong evidence that establishment and maintenance of polarity in NPCs is critical to normal cortical development.

Studies in MDCK cells have identified 3 complexes (Crb3/PALS1/PATJ, Par3/Par6/aPKC and MALS/PALS1) that are critical for maintaining polarity (Margolis and Borg, 2005). MALS (also known as Lin-7 or Veli) is a PDZ domain-containing protein that is of particular interest because it plays a role in apico-basal polarity and also specifies cell fates in C. elegans by controlling the basal localization of the EGF receptor LET-23 in vulval precursor cells (Kaech et al., 1998). LET-23 is mislocalized to the apical membrane in lin-7 mutants, leading to a mis-specification of vulval cell fates (Kaech et al., 1998). Thus, MALS has a critical role in cell fate specification during invertebrate development, making it an attractive candidate gene that might regulate cell fate of NPCs. In vertebrates, there are three MALS genes, MALS-1, -2 and -3. Silencing MALS-3 in MDCK cells resulted in defects in tight junction formation and the loss of several binding partners of MALS-3 such as PALS1 (Margolis and Borg, 2005; Olsen et al., 2005a). PALS1 forms a complex with Crb3 and PATJ and can also interact with Par6, thus linking the Par6 signaling complex to other apically localized proteins such as Crb3 and MALS-3 (Roh et al., 2002). Together, these proteins are positioned apically to link transmembrane signaling proteins with cytoskeletal structures and/or cell nuclei (Roh and Margolis, 2003).

Despite progress in understanding the role of MALS proteins in MDCK cells in vitro, much less is known about their role in vivo. MALS-1 and -2 are expressed at synapses (Misawa et al., 2001). However, MALS-1/2 double knockout mice did not reveal obvious synaptic defects due to an upregulation of MALS-3 in the mice (Misawa et al., 2001). Analysis of mice in which all three MALS genes were disrupted revealed that MALS proteins play a critical role in presynaptic vesicle recycling; the triple knockout (TKO) resulted in reduced synaptic transmission and neonatal lethality (Olsen et al., 2005a; Olsen et al., 2006).

Here we explore the role of MALS-3 in NPCs. We show that MALS-3 is localized to the apical surface of progenitors during neurogenesis, where it interacts primarily with PALS1 and CASK (also known as Lin-2). Analyses of MALSTKO embryos reveal that MALS-3 is required for the normal regulation of NPC proliferation and differentiation at the onset of neurogenesis. Furthermore, MALS is required for the continued apical localization of PATJ and PALS1 proteins in NPCs. Collectively these data suggest that MALS plays a role in maintaining polarity complexes in NPCs and that loss of MALS affects early neurogenesis. Thus, although MALS protein is evolutionarily conserved, it appears to control polarity rather than cell fate in mammalian corticogenesis.

Materials and Methods

In situ hybridization and immunohistochemistry

In situ hybridization was performed as described previously (Chen et al., 2005). Polyclonal antibodies were generated by immunizing rabbits (BAbCo Laboratories, Berkeley, CA) with full length MALS-3 or Mint protein in BL21 pLysS (Stratagene, La Jolla, CA). MALS-3 antibodies were affinity-purified from the sera of immunized rabbits using MALS-3-GST fusion proteins coupled to Affi-Gel 10 beads (Bio-Rad Laboratories, Hercules, CA). Immunohistochemistry was performed as described in (Cappello et al., 2006), and other antibodies used are listed in Supplementary Table 1. Secondary antibodies used for immunoblots were anti-mouse Alexa Fluor® 680 (Molecular Probes, Eugene, OR) and anti-rabbit IRDye® 800 (Rockland Immunochemicals, Gilbertsville, PA) (1:30,000). Secondary antibodies used for immunostaining were Cy-5 anti-rabbit and Texas Red anti-mouse (Jackson ImmunoResearch Laboratories, Westgrove, PA) (1:500). F-actin was visualized using Texas Red conjugated phalloidin (Molecular Probes, Eugene, OR) (1:50) and nuclei were visualized by SYTO11 (Molecular Probes) (1:1000).

Cell fractionation, immunoblot imaging and data analysis

Details of sample preparation are described in Supplementary Methods. Continuous density gradient centrifugation was performed as described earlier (Vogelmann and Nelson, 2005). Proteins from samples were separated by SDS PAGE (Laemmli, 1970). Blots were processed as described in the Western Blot protocol for the Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE). Three such gradients were used to analyze the density profile of the various proteins described in this study, and three additional gradients were used for immuno-precipitation. Raw images of blots were generated using Odyssey software and processed using Adobe Photoshop. Quantification of protein present in each protein band was based on the integrated intensity above background of the area of the blot containing the band as described in the Odyssey Infrared Imaging System user guide (http://www.licor.com). Graphs of protein distribution throughout density gradients were generated using Microsoft Excel. Integrated intensity data and protein concentration for each set of fractions were converted linearly to arbitrary units from zero to 100, with 100 being the maximum value for each data set.

In utero electroporation

In utero electroporation was performed as described (Kawauchi et al., 2003). Embryos were collected 2 or 5 days post-surgery for analysis; 5 embryos were analyzed for each time point.

Labeling index and quit fraction

For labeling index (LI) studies, pregnant mice were injected with BrdU at E11.5 or E13.5, and embryos were collected 2 hours later. The LI is the ratio of cells in the VZ that incorporated BrdU divided by the total number of nuclei in the field and represents the fraction of cells in S-phase during the BrdU pulse. To calculate a modified labeling index, we calculated the ratio of BrdU+ cells over the total number of cycling cells (Ki67+), which provides a relative measure of cell cycle length. For quit fraction analyses, pregnant mice were injected with BrdU at E12.5 and embryos were collected 24 hours later (N=3 for each analysis).

Results

Expression of MALS in progenitor cells of the developing neocortex

To explore the function of MALS in cortical development, we first assessed the expression and localization of MALS in the rat neocortex. RT-PCR and in situ hybridization revealed that while MALS-3 is strongly expressed by NPCs of at E15.5, MALS-1 and -2 are not detected in the VZ (Suppl. Fig. 1). Immunohistochemistry revealed that MALS-3 protein is localized to the apical domain of NPCs lining the lateral ventricle (Fig. 1J-L) and in the apical domains of a variety of other cell types throughout the body (Suppl. Fig. 2). We next characterized the temporal expression of MALS-3 and found that MALS-3 is expressed diffusely throughout the apico-basal extent of NPCs as early as E9.5 in VZ cells (Fig. 1D-F). By E11.5, however, MALS-3 staining is localized apically at the ventricular surface (Fig. 1G-I), where it is maintained throughout neurogenesis (Fig. 1J-L).

Figure 1
MALS-3 is expressed by and localizes to the apical surface of neural progenitors during neurogenesis

When we compared the localization of MALS with that of F-actin (concentrated near adherens junctions at the boundary between the cells’ basolateral and apical regions: Chenn et al., 1998), we found that MALS-3 occupies a domain that is largely supra-apical to that of F-actin (Fig. 1E). In en face views of the ventricular surface, it is apparent that F-actin staining forms rings around the apical endfeet of progenitor cells (Fig. 1C). MALS-3 overlaps partially with these F-actin rings, but also stains regions that are both more apical and continuous (Fig. 1C), suggesting that MALS-3 protein is distributed in a cup-like pattern at the apical ends of NPCs.

Comparison of MALS-3 localization with that of other apically-localized proteins (ZO-1, ZO-2, pan-cadherin, β-catenin, PALS1 and Crb3) (Calegari et al., 2005; Cappello et al., 2006; Imai et al., 2006) revealed partially or entirely overlapping expression domains at the apical surface (Fig. 1A,B; Fig. 2). Of particular interest was the overlapping expression with PALS1 (Fig. 2G-I), a protein that directly binds to MALS-3 via an L27 interaction domain. Thus, MALS3 and several proteins with which it can interact biochemically with are expressed in and localized to the apical domains of NPCs during neocortical development.

Figure 2
Localization of MALS immunoreactivity compared with other proteins in the VZ

MALS-3 is associated with cell membranes

Although MALS-3 does not contain a transmembrane domain, in other systems, it associates with membranes via interactions with other proteins (Roh and Margolis, 2003; Straight et al., 2006). To ascertain whether the MALS-3 in VZ cells is associated with the plasma membrane, we used high speed centrifugation and density gradient separation to separate membranes and membrane-associated proteins from the cytosol. High speed centrifugation revealed that MALS-3 exists in two pools in the telencephalon: approximately 2/3 of the total MALS-3 was present in the cytosolic fraction (S100), whereas roughly a third was distributed between Triton-soluble (TX100) and Triton-insoluble (SDS) membranes (Fig. 3A). This suggests that a substantial fraction of the MALS-3 in NPCs is associated with cell membranes. To compare the distribution of MALS-3 with that of other proteins with which it might potentially interact, we probed the same fractions with antibodies against CASK, Mint (also known as Lin-10), and PALS1, as well as with antibodies to proteins are interesting in the context of apico-basal polarity and adherens junctions. These studies revealed that CASK is associated primarily with Triton-insoluble membranes, whereas Mint was found mostly in the cytosol (Fig. 3A). PALS1, Dlg, β-catenin, Cadherin, and the Na+/K+ ATPase were found primarily in membrane fractions, whereas p38γ/SAPK3 was found almost exclusively in the cytosol (Fig. 3A).

Figure 3
Distribution of proteins between membrane and cytosol in postnuclear supernatant from the embryonic telencephalon

To further refine the localization of and possible associations between MALS-3 and other polarized proteins, we performed density gradient centrifugation of homogenates from the embryonic rat telencephalon followed by a quantitative analysis of proteins present in each fraction following Western blot analysis (Vogelmann and Nelson, 2007). Markers for distinct sub-cellular compartments were utilized to distinguish fractions containing cytosol, Golgi membranes, and plasma membranes (Suppl. Fig. 4). The signal intensities of bands in Western blots were normalized to the maximal value obtained for each protein and then plotted to represent the relative amount of protein in each fraction from the gradient (Suppl. Fig. 4). These experiments revealed that, as predicted from high-speed centrifugation, MALS was found both in cytosolic fractions (peak at fraction 17) and in fractions containing plasma membrane (fractions 5 and 14) (Fig. 3B,C). MALS and CASK shared nearly identical distributions, each showing an overall maximum at fraction 17 and local maxima at fractions 5 and 14 (Fig. 3B,C). This distribution suggests that CASK and MALS are each found in both the cytosolic and plasma membrane pools, consistent with the possibility that these two proteins interact biochemically in NPCs. Mint was distributed in a single peak centered at fraction 17, suggesting that Mint is primarily cytosolic in VZ cells. Thus, in contrast to the close association between LIN-7, LIN-2, and LIN-10 in C. elegans, only MALS and CASK localize to plasma membranes in NPCs (although all three proteins might still interact in the cytosol).

To identify other potential MALS interactors, we examined the density distributions of 10 proteins that have been implicated in cell polarity and/or the formation of apically localized junctions. Collectively these showed diverse behaviors during gradient centrifugation (Fig. 3D,E), but several were distributed across the gradient in patterns similar to that of MALS. PALS1, which interacts with MALS and is required for tight junction formation in MDCK cells (Hurd et al., 2003; Roh et al., 2002; Straight et al., 2004), exhibited a major peak at fraction 14 and a minor peak at fraction 5, as well as a broad distribution across the cytosolic fractions. It is likely that MALS and PALS1 proteins interact biochemically, since PALS1 could be co-immunoprecipitated with antibodies to MALS-3 (Suppl. Fig. 3C). Interestingly, α-catenin, β-catenin, and cadherin were similarly distributed in density gradients (Fig. 3D,E), suggesting that each is associated with the plasma membrane.

The junctional proteins ZO-1 and ZO-2 shared a major plasma membrane peak at fraction 14, but only ZO-2 had a second peak in the cytosol, at fraction 17 (Fig. 3D,E). Dlg, which is commonly considered to be basolaterally localized but is found at tight junctions in MDCK cells (Albertson and Doe, 2003; Peng et al., 2000), exhibited peaks of intensity in plasma membrane fractions 5 and 14 and cytosolic fraction 18. In addition, Dlg could be co-immunoprecipitated with MALS-3 from these gradients (Suppl. Fig. 3C), consistent with recent reports that Dlg1 interacts with all three MALS proteins via associations with the PALS1 family member MPP7 (Bohl et al., 2007). The polarity proteins Par6A and Par6B were found primarily in the cytosolic fractions, although Par6B also displayed a smaller peak in the plasma membrane, in fraction 5. Interestingly, previous studies have suggested that Par6B localizes to the cytosol in MDCK cells, whereas Par6A is associated with the tight junction protein ZO-1 (Gao and Macara, 2004). However, these relationships were not apparent from the density gradients under study here. Finally, PATJ was found almost exclusively in fraction 18 within the cytosolic pool (Fig. 3D). This is surprising because PATJ associates with tight junctions in MDCK cells (Shin et al., 2005), where it interacts with PALS1 (Roh et al., 2002; Straight et al., 2006) and the transmembrane protein Crumbs (Makarova et al., 2003).

In sum, of the potential MALS interactors studied here, CASK showed a distribution in the density gradients that was nearly identical to that of MALS, and the distribution of PALS1 was strikingly similar in lighter, plasma membrane fractions. These observations are consistent with evidence that CASK and PALS1 interact with MALS in MDCK cells (Straight et al., 2006). In addition, analysis of homogenates from the postnatal brain, in which MALS-1 and -2 proteins are associated with synapses, has revealed that MALS associates with CASK, Mint and Liprins in postmitotic neurons (Olsen et al., 2005a).

MALS triple knockout mice do not display gross morphological abnormalities of the developing neocortex

Based on its expression pattern and biochemistry, we hypothesized that MALS-3 might play a critical role in either establishing and/or maintaining apical polarity early in neurogenesis. To test this hypothesis, we examined corticogenesis in mice in which all three MALS genes have been genetically disrupted (Olsen et al., 2005a). Mice that lack MALS-1 and -2 appear normal, due to the upregulation of MALS-3 in neurons, and were therefore used as controls in our study. Mice in which only MALS-3 is mutated also lack obvious defects, presumably due to compensation by the two other MALS isoforms (Misawa et al., 2001). MALS triple knockout (MALSTKO) mice were bred and genotyped as described earlier (Olsen et al., 2005a). MALSTKO pups die soon after birth (Olsen et al., 2005a), and examination of their brains at P0 revealed no obvious morphological abnormalities such as smaller brain size or defects in cortical lamination (as evidenced by immunostaining with layer specific antibodies: Suppl. Fig. 5).

MALS-3 is required for the maintenance of apical polarity in NPCs

Although it comprises less than 2% of the entire membrane surface (Huttner and Kosodo, 2005; Kosodo et al., 2004), the apical domain of NPCs represents a region that is actively targeted by several proteins, including signaling molecules such as β-catenin and aPKC (Chenn and Walsh, 2002; Chenn et al., 1998; Imai et al., 2006) and the epidermal growth factor receptor EGFR ((Sun et al., 2005). Indeed, the MALS homolog LIN-7 plays an essential role in the basolateral targeting of EGF receptors in C. elegans vulval precursors (Kaech et al., 1998), thus we examined EGFR localization in MALSTKO brains. However, we observed no differences between controls and mutants (data not shown). Since MALS-3 regulates apico-basal polarity in MDCK cells, we hypothesized that MALS-3 might play a similar role in NPCs (Straight et al., 2006). At E13.5, the VZ appeared intact in MALSTKO brains, although MALS immunoreactivity was absent from the ventricular surface as expected (Fig. 4A,B). The MALSTKO mutants also showed a complete loss of PATJ staining (Fig. 4H), which is normally concentrated apically (Fig. 4G). The localization of other apical proteins was unaltered (Fig. 4 and data not shown), including that of PALS1 (Fig. 4C,D), which interacts directly with MALS in MDCK cells (Kamberov et al., 2000). The loss of PATJ staining by E13.5 was surprising since MALS-3 associates with PATJ through its interaction with PALS1, a part of the Crb3/PALS1/PATJ complex. Moreover, the density centrifugation experiments showed distinct patterns for MALS-3 and PATJ, suggesting that they occupy distinct intracellular domains (Fig. 3). Nevertheless, PATJ was completely absent from the apical surface of MALSTKO NPCs.

Figure 4
MALS is required for the maintenance but not establishment of apical localization of PALS1 and PATJ during corticogenesis

Similar analyses at E18.5 revealed that, in addition to the continued loss of PATJ at the apical surface of NPCs (Fig. 4O,P), PALS1 immunoreactivity was also lost from the apical domain in MALSTKO mutants (Fig. 4K,L). This suggests that MALS-3 is not required for the initial apical targeting of PALS1 in NPCs but is important for maintaining its apical localization. Although in many mutants the distribution of Crb protein appeared normal (Fig. 4M,N), we occasionally observed a reduction in the intensity of apical Crb staining (data not shown). Because the antibody used to detect Crb3 also detects Crb2 (unpublished data, O. Olsen and K. Srinivasan), it is possible that changes in the localization of one isoform might be masked if the other were unaltered. No changes were observed in the distributions of Par3, Par6, or aPKCζ (Fig. 4I,J and data not shown), suggesting that MALS does not affect the Par6 signaling pathway in NPCs. It was interesting to note that despite the loss of at least 3 proteins (MALS-3, PATJ, PALS1, and occasionally Crb) from the apical surface, the VZ still appeared intact at E18.5, suggesting that none of these proteins are crucial for maintaining the structural integrity of the VZ. Western blots revealed no obvious changes in the total protein levels of PALS1 or PATJ (Fig. 4Q), suggesting that MALS is required only for the apical localization and not the overall stability of PALS1 and PATJ in NPCs. These data thus contrast with recent studies in which the silencing of MALS-3/Lin-7 in MDCK cells resulted in a loss of PATJ from tight junctions due to alterations in protein stability (Straight et al., 2006).

Adherens junctions remain intact in MALSTKO brains

In MDCK cells, the loss of PATJ by RNAi-mediated gene silencing leads to a loss of tight junctions, as revealed by the loss of ZO-1 expression at these structures (Shin et al., 2005). Although NPCs in vivo do not form tight junctions (Aaku-Saraste et al., 1996), they do form adherens junctions at which ZO-1 and ZO-2 co-localize. The intact appearance of the VZ in MALSTKO embryos suggested that adherens junctions were maintained in the mutant cortex. This was confirmed by the continued normal expression of ZO-1 and ZO-2 in mutant and control embryos (Fig. 5A,B and data not shown). We also assessed the localization of cadherins, which form an integral component of adherens junctions, and the signaling protein β-catenin, since changes in their localization would likely have profound consequences on NPCs (Chenn and Walsh, 2003). Again, no differences in protein localization were observed in MALSTKO and control cortices (Fig. 5C-F). Collectively these results suggest that MALS is not required for the establishment or maintenance of adherens junctions in the developing brain.

Figure 5
Adherens junction integrity is not compromised in MALSTKO embryos

Analysis of NPC proliferation and differentiation in MALSTKO mice

To ascertain whether the loss of MALS-3 and subsequently PATJ and PALS1 altered the process of neurogenesis, we immunostained brains with antibodies that distinguish NPCs (nestin), neurons (Tuj1, NeuN and Tbr1), and glial cells (GFAP) (Fig. 5 and data not shown). At E13.5, staining for nestin and GFAP appeared similar in mutant and control mice (Fig. 5G,H). However, we observed a broadening of the TuJ1 expression domain in MALSTKO mutants compared to controls (Fig. 5I,J), suggesting that the production of neurons might be expanded or initiated prematurely in MALS mutants.

To assess this change in neurogenesis more quantitatively, we first ascertained whether the loss of MALS altered cortical NPC proliferation. BrdU was injected at E11.5 or E13.5, embryos were collected 2 hours later, and the fraction of VZ cells that were BrdU+ was used to calculate the labeling index (LI, representing cells in S phase). The LI at E11.5 was significantly lower in MALSTKO mice (0.36±0.05) compared to controls (0.44 ± 0.03, p < 0.03) (Fig. 6A). A reduction in LI suggests that either fewer VZ cells were cycling in the mutant VZ, or the same number of cells were cycling more slowly. The limited availability of MALSTKO mice made it impossible to perform cumulative labeling studies to ascertain the total length of the cell cycle. Thus, to assess BrdU incorporation only in actively cycling cells, we immunostained embryos for both BrdU and Ki67 (which is expressed by all cycling cells). If the change in LI were due to a decreased number of cycling cells, we should see no difference in fraction of Ki67+ cells that were also BrdU+ in mutants. However, if the decrease in LI arose from a lengthened cell cycle, fewer BrdU+ cells in the Ki67+ population would be expected (Chenn and Walsh, 2003; Siegenthaler and Miller, 2005). In fact, we observed a significant decrease in the fraction of Ki67+ cells that were BrdU+ (control: 0.80 ± 0.04; MALSTKO: 0.63 ± 0.06; p<0.01), suggesting that MALS deficient progenitors cycle more slowly than do control NPCs.

Figure 6
MALSTKO embryos display significant differences in labeling index and quit fraction during early neurogenesis

At early stages of corticogenesis, wild-type NPCs divide rapidly and most divisions are symmetric, producing more progenitors; later, the cell cycle slows and the fraction of daughter cells that differentiate into neurons increases (Calegari et al., 2005). The lower labeling index in E11.5 MALSTKO brains and the broadened domain of Tuj1 immunoreactivity suggested that early NPCs might have prematurely entered the neurogenic phase of development. To assess the fraction of cells that differentiated following early divisions in MALSTKO mutants, embryos were injected with BrdU at E12.5, collected 24 hrs later, and the fraction of BrdU+ cells that had lost Ki67 staining was used to calculate the Quit Fraction (QF) of cells that had exited the cell cycle. The QF in MALSTKO embryos (0.53 ± 0.03) was significantly higher than that observed in littermate controls (0.45 ± 0.03, p<0.03) (Fig. 6B), demonstrating that MALSTKO progenitors differentiate prematurely during early stages of neurogenesis. Since the orientation of the mitotic spindle can affect the outcome of NPC divisions (Chenn and McConnell, 1995; Fish et al., 2006), we asked whether the shift towards more neurogenic divisions might be correlated with changes in spindle orientation. We assessed the orientations of VZ cells in anaphase of the cell cycle at E11.5 and found no significant differences between control and MALSTKO embryos (data not shown), suggesting that MALS3 does not regulate neurogenesis by controlling the orientation of mitotic spindles. Interestingly, MALSTKO progenitors appeared to catch up with their control counterparts by E13.5. BrdU injections at this age produced no differences in LI of MALSTKO mice compared to controls (Fig. 6C). Thus, although the loss of MALS-3 alters NPC proliferation during early stages of neurogenesis, by mid-neurogenesis NPCs resumed normal cell cycle dynamics. We presume that this recovery explains the relatively normal size of the brain and cerebral cortex in newborn MALSTKO animals.

Deliberate mistargeting of MALS-3 results in a breakdown of VZ

To ascertain whether the apical localization of MALS is required for its function, MALS-3 was tagged with an N-terminal myristoylation sequence (MGSSKSKPKDPS) (CA-myr_MALS-3) and co-electroporated with CA-EGFP into the lateral ventricles of E13.5 embryos. As a control, full length MALS-3 without a myristoylation tag (CA-FL_MALS-3) was electroporated into embryos in the contralateral uterus. In addition, to eliminate the possibility that the expression of any myristoylated protein would disrupt VZ cells, we electroporated embryos with a construct expressing myristoylated Crb3 (CA-Myr_Crb3). At E18.5, embryos electroporated with FL_MALS-3 appeared normal, with large numbers of EGFP+ neurons and their axons present in the cortical plate (Suppl. Fig. 6A). We observed no change in the localization of Crb3, PALS1 and aPKC in the VZ in these brains (Fig. 7A-C). Similarly, introduction of myr_Crb3 had no observable effect on the structure of the VZ or the localization of the apically localized proteins ZO-1, PATJ, and PALS1 (Fig. 7D,G,J), even though Crb protein itself was observed in the basolateral regions of EGFP+ cells (Fig. 7M).

Figure 7
The mislocalization of MALS-3 disrupts polarity

In contrast, embryos electroporated with Myr_MALS-3 showed gross morphological abnormalities (Fig. 7P-R; Suppl. Fig. 6C). The integrity of VZ was compromised, and the lateral ventricles were infiltrated with TuJ1 positive cells (Fig. 7R). Within 2 days after electroporation, at E15.5, breaks were observed in the normal apical staining patterns of ZO-1 (Fig. 7E,F), PATJ (Fig. 7H,I), PALS1 (Fig 7K,L) and Crb (Fig 7N,O) near EGFP+ cells, suggesting that adherens junctions had been disupted at these sites. Consistent with this interpretation, we also observed breaks in β-catenin staining in the VZ (Suppl. Fig. 6C). Misplaced cells that were positive for nestin were also seen in the ventricle at E15.5 (although in smaller numbers compared to E18.5), suggesting they were delaminated NPCs (data not shown). These phenotypes are reminiscent of the conditional loss of β-catenin in the developing forebrain, in which adherens junctions integrity is compromised, leading to a delamination of NPCs and appearance of ectopic cells in the lateral ventricles (Junghans et al., 2005). Interestingly, we did not observe any changes in the localization of β-catenin in MALSTKO mutant brains (Figs. (Figs.4;4; 5C,D), suggesting that MALS-3 is not normally required for the apical localization of β-catenin. These observations suggest that the overexpression of myr_MALS-3 in NPCs disrupts global apico-basal polarity and affects junctions secondarily, rather than specifically interfering with β-catenin localization.

Discussion

MALS-3 is expressed and localizes apically within NPCs in the embryonic VZ. We show that MALS-3 associates with cell membranes and interacts biochemically with CASK, Mint and PALS1 during corticogenesis. The genetic disruption of all three MALS genes in mice leads to an early loss of PATJ protein from the apical domain of NPCs, followed by a loss of PALS1 at later stages of neurogenesis. These findings suggest that MALS-3 is required for maintaining the apical localization of the Crb3/PALS1/PATJ complex. We observed no changes in adherens junction proteins such as ZO-1 or ZO-2, or of key signaling proteins such as aPKCζ or β-catenin, suggesting that adherens junctions remained intact throughout corticogenesis. The loss of MALS also resulted in a significant slowing of the cell cycle and premature differentiation during early (but not later) stages of neurogenesis. These differences, however, did not visibly alter in the overall construction of the cerebral cortex, which had a normal appearance at P0. Finally, we found that the overexpression of a myristoylated form of MALS severely disrupted the apico-basal polarity of NPCs. Collectively these data suggest that MALS is required for the maintenance of apico-basal polarity and the normal control of proliferation in the developing forebrain.

MALS-3 is localized supra-apical to F-Actin and associates with CASK, Mint, and PALS1

The MALS homolog LIN-7 plays an essential role in the basolateral targeting of EGF receptors in c.elegans vulval precursors (Kaech et al., 1998). In contrast, in mammalian NPCs, MALS is localized apically, as is EGFR (Sun et al., 2005). However, MALS does not appear to play a critical role in targeting EGFR apically in NPCs, since the distribution of EGFR was not obviously altered in mutants. This is perhaps not surprising in light of previous observations showing that EGFR is expressed during and plays important functions at later stages of cortical development (Sun et al., 2005) compared to the earlier role for MALS demonstrated here.

It appears that the subcellular localization of MALS proteins is tissue-dependent in vertebrates. For example, renal epithelial cells express all three MALS proteins, but their subcellular localizations vary among different cell types. In intercalated cells of the kidney, MALS-1 localizes to the cytosol, whereas in the collecting ducts, MALS-3 can be found in predominantly basal or in apical locations (Olsen et al., 2005b). In MDCK cells, MALS-3/Lin-7c, PALS1, and Crb3 all localize to tight junctions, which form a boundary between the cell’s apical and basolateral domains (Roh and Margolis, 2003). It is not yet clear why MALS-3 and its partners show such variability in their localization patterns in different cell types; one possibility is that MALS-3 becomes localized to regions of cell-cell contact, including tight junctions in epithelial cells and adherens junctions in NPCs. However, in the latter cell type, MALS-3 clearly occupies a domain that is supra-apical to that of F-actin and adherens junctions, suggesting that an active sorting machinery directs MALS-3 into this location. PALS1 is unlikely to mediate this localization because MALS-3 expression was not altered in MDCK cells silenced for PALS1(Straight et al., 2004). One possibility is thatβ-catenin mediates the junctional localization of MALS-3, since β-catenin appears to be involved in actively localizing MALS-3 from the cytosol to calcium-mediated cadherin junctions in MDCK cells and to early synapses in hippocampal neurons (Perego et al., 2000). However, MALS-3 is not required for the apical localization of β-catenin since normal expression patterns of β-catenin were observed in MALSTKO mutants.

Irrespective of localization of MALS, many of its biochemical partners appear to be highly conserved. In MDCK cells, MALS-3 plays a role in stabilizing the PALS1 complex at tight junctions; cells silenced for MALS-3 display a loss of apical localization of PALS1 and ZO-1, resulting in a delay in the formation of tight junctions (Straight et al., 2006). This phenotype was fully rescued by introduction of exogenous PALS1, providing strong evidence that MALS-3 function is mediated by PALS1 and that MALS-3 maintains the apical localization of PALS1. This interaction is not reciprocal, since MALS-3 expression is maintained in the absence of PALS1 (Straight et al., 2004). Consistent with these previous studies, MALSTKO animals also showed a loss of PALS1, but only at later stages of neurogenesis. This suggests that MALS-3 is not required for initial localization of PALS1 but for maintaining PALS1 apically in NPCs. We observed an earlier loss of PATJ protein from the apical surfaces of VZ cells, an observation that was surprising in the absence of direct biochemical interactions between these two proteins. Surprisingly, the loss of MALS did not appear to affect the expression levels of these proteins as assessed in Western blots, suggesting that MALS-3 is primarily involved in maintaining the apical localization of the PALS1 complex. In contrast, MDCK cells silenced for Lin-7c (MALS3) displayed a loss of both PALS1 and PATJ due to rapid protein turnover. The authors speculate that Lin-7c is required for stabilizing PALS1 protein at the apical surface. Since PATJ interacts with PALS1, the loss of PALS1 might result in the loss of PATJ from these cells (Straight et al., 2006). However, our data from MALSTKO mutant brains suggest that the role for MALS in apical protein localization can be separated from its role in protein stabilization.

In contrast to the role of MALS in maintaining polarity, PALS1 appears to play a critical role in establishing apico-basal polarity. In MDCK cells, PALS1 is required not only for the formation of tight junctions, but also for that of adherens junctions, which normally adopt a position subapical to tight junctions (Wang et al., 2007). In PALS1-silenced MDCK cells, the formation of adherens junctions was severely disrupted due to the loss of E-cadherin at the apical surface (Wang et al., 2007). These observations, together with data from MDCK cells silenced for Lin-7/MALS-3 (Straight et al., 2006), provide strong evidence that PALS1 functions upstream of MALS-3, and that PALS1 can compensate to a large extent for the loss of MALS-3 in cell polarization. Since we did not observe a loss of PALS1 from the apical surfaces of NPCs until later stages of corticogenesis, we suspect that PALS1 was compensating functionally for the loss of MALS-3. Based on these observations, we would predict that the loss of PALS1 in NPCs should generate a more profound disruption of corticogenesis than the loss of all three MALS genes.

NPC proliferation and differentiation depend on apico-basal polarity but not the integrity of adherens junctions during cortical development

The relationship between apico-basal polarity and cell-cell junctions in controlling NPC proliferation has been a subject of recent interest, particularly in light of the role of junctions in mediating intercellular signaling. In Drosophila, intact junctions are essential for the normal proliferation of NPCs (Lu et al., 2001); however, this requirement appears to be less strict during vertebrate neurogenesis. For example, the genetic disruption of the Rho family GTPase Cdc-42 results in a loss of adherens junctions and the mislocalization of mitotically active progenitors from the apical into the basal region of the cortical VZ; however, the misplaced cells continued to cycle despite their aberrant positions (Cappello et al., 2006). In another study, conditional mutations in aPKCλ (which encodes a key component of the apically localized PAR complex) produced a complete loss of adherens junctions yet failed to disrupt neurogenesis, which proceeded normally (Imai et al., 2006). These observations suggest that the maintenance of adherens junctions is not absolutely required for the normal regulation of NPC proliferation.

In the MALSTKO mutants, we observed a complementary situation. First, adherens junctions appeared intact in the mutant brain, demonstrating that MALS-3 is not required for the integrity of adherens junctions. However, we observed significant abnormalities in both proliferation and differentiation of NPCs. First, MALSTKO mutants exhibited a lower LI and a lengthening of the cell cycle at the onset of corticogenesis, although proliferation recovered a few days later. Second, MALSTKO mutants displayed premature neuronal differentiation at early times during neurogenesis. Interestingly, these alterations were also temporary: the cortex as a whole appeared relatively normal at P0, and MALSTKO mutants failed to display any gross morphological changes such as smaller brain size or loss of select neuronal populations at birth.

Although the transience of the changes in proliferation presents a puzzle, we suspect that this may reflect the fact that, in normal animals, the transition from diffuse basolateral to strictly apical localization of MALS-3 occurs at ~E11.5, the same time at which abnormalities in proliferation were observed. We hypothesize that early NPCs might be particularly vulnerable to alterations in the molecular composition of their apical complexes, and that at slightly later stages, the presence of other apical polarity proteins might restore normal patterns of cell proliferation. We further hypothesize that the higher QF observed at E13 might be the result of the earlier shift in LI. Previous studies have suggested that lengthening the cell cycle in NPCs can cause a shift from proliferative divisions to neuron-generating divisions (Calegari et al., 2005). Thus, the more slowly cycling progenitors in E11 MALSTKO mutants might differentiate into neurons earlier than their control counterparts once they complete their cell cycle, thus generating a higher QF by E13.

In some mutant mouse lines, higher QFs can be accompanied by a concomitant loss of NPCs, which depletes the progenitor pool and results in smaller brain sizes due to a loss of later-born neurons and/or glial cells. For example, loss of Brg1, an ATP-dependent chromatin remodeling protein, leads to precocious differentiation, the premature depletion of NPCs prior to gliogenesis, and a severe loss of oligodendrocytes and glial cells (Lu et al., 2001). Gliogenesis, however, appeared normal in MALSTKO mice when compared to controls, further emphasizing that the consequences of altered QFs at E13.5 were subtle, most likely because of the rapid recovery in NPC proliferation.

It is not yet clear how the loss of MALS causes a shift towards neurogenesis in cycling cells at early stages of cortical development. One possibility is that MALS-3 might be important for the asymmetric distribution or localization of a cell fate determinant that regulates NPC proliferation and/or differentiation. For example, in epithelial cells, the tight junction protein ZO-1 influences the G1/S-phase transition by sequestering a complex formed by CDK4 and the transcription factor ZONAB away from the nucleus in the cytoplasm (Balda et al., 2003; Sourisseau et al., 2006). It is plausible that the disruption of MALS proteins in NPCs may disrupt the localization of similar cell cycle regulators and thus lead to premature cell cycle exit, although the molecular mechanisms underlying this phenomenon are as yet unknown.

Deliberate mistargeting of MALS-3 results in a breakdown of VZ integrity

Although a complete loss of MALS-3 did not affect the structural integrity of the VZ or intercellular junctions, the overexpression of MALS-3 tagged with an N-terminal myristoylation sequence resulted in profound alterations in the localization of PALS1 and PATJ, as well as a loss of adherens junction proteins from the lumenal surface. Many electroporated cells delaminated from the VZ and spilled into the lateral ventricles, suggesting that the integrity of adherens junctions was profoundly compromised by the mislocalization of MALS-3. We suspect that the mislocalization of MALS-3 affects the localization of other proteins, particularly its direct binding partner PALS1, as well as that of other apical proteins such as aPKC and PAR proteins, which require PALS1 for their normal localization (Margolis and Borg, 2005). Loss of these proteins from the apical surface might trigger the dissolution of adherens junctions (Imai et al., 2006), which could leads to delamination of NPCs. However, MALSTKO mutant brains and normal brains electroporated with FL_MALS-3 showed no alteration in aPKC staining, suggesting that MALS-3 is not normally required for the localization of the PAR3/PAR6/aPKC complex. Collectively these results suggest that while a loss of MALS-3 is tolerated by NPCs, its mislocalization is not, with the latter causing far more detrimental effects on apical polarity than the loss of the MALS protein.

Supplementary Material

Suppl. Fig. 1

Supplementary Figure 1: Detection of gene products in the E14 rat dorsal telencephalon. (A) Reverse-transcriptase PCR detection of RNA transcripts of MALS-1, MALS-2, MALS-3, CASK, Mint and PALS1 in poly-adenylated RNA purified from rat E14 dorsal telencephalon (Telen, +RT) and P3 cerebellum (Cerebellum, +RT). Control reactions without reverse transcriptase were run in parallel (−RT). (B) In situ hybridization to detect MALS-1, MALS-2, and MALS-3 mRNAs in sections of E14 rat dorsal telencephalon (scale bar 100 μm). MALS-3 appears to be the predominant MALS isoform expressed in VZ cells. C) Western blot detects MALS3, MALS2A and 2B and MALS1 at the appropriate molecular weight in E14 rat telencephalon. Abbreviations: pial surface (P), preplate (PP), ventricular zone (VZ), cerebral ventricle (V).

Suppl. Fig. 2

Supplementary Figure 2: Localization of MALS-3 immunoreactivity in rat tissues at E14. Sections are stained to reveal MALS-3 (green), F-actin (red) and cell nuclei (blue). (A-C) MALS-3 immunoreactivity at the apical surface of VZ cells is shown to facilitate comparison with other organs from the same embryo. (D-F) No distinct polarization of MALS-3 is seen in the developing eye. (G-O) MALS-3 localizes to the lumenal surface in the oral cavity (G-I), embryonic lung (J-L) and embryonic gut (M-O). Scale bar = 100 μm. Abbreviations: pial surface (P), ventricular zone (VZ), cerebral ventricle (V), lens (L), retina (R), retinal pigmented epithelium (RPE), epithelia of the gut and oral cavity (E), lumen of the gut and oral cavity (L), and lumen of the bronchial buds (carets).

Suppl. Fig. 3

Supplementary Figure 3: Distribution of proteins between membrane and cytosol in telencephalon postnuclear supernatants. The dorsal telencephalon was dissected from E14 rat embryos and homogenized in CSK homogenization buffer (50 mM NaCl, 150 mM sucrose, 10 mM Pipes, pH6.8, 3 mM MgCl2 with 1X Sigma protease inhibitor) by sonication. Cellular debris was removed by centrifugation at 1000 × g for 10 minutes at 4°C. High-speed centrifugation was used to pellet membranes and membrane-associated protein from PNS as well as to separate Triton-X100-soluble material from Triton-X100-insoluble material in the membrane pellet. The dorsal telencephalon from 10 E14 embryos was homogenized in 2 ml of CSK or 2 ml of CSK without Mg+2 supplemented with EDTA. 1 ml of PNS was centrifuged at 100,000g for 40 minutes to pellet membranes. The supernatant (S100) was removed and the pellet was resuspended in an equal volume of CSK with 0.5% Triton X-100 at 4°C and centrifuged again at 100,000 × g to pellet Triton insoluble material. The supernatant (TX100) was removed and the pellet from this spin was resuspended in 2 ml of SDS PAGE sample buffer while the previous supernatants were brought to 2 ml with 2× SDS PAGE sample buffer (Vogelmann and Nelson, 2007). (A) Triton soluble membranes (TX100), triton insoluble membranes (SDS) and cytosol (S100) from E14 rat telencephalon homogenate postnuclear supernatant (PNS) with either Magnesium ion (Mg++ group) or chelating agent (EDTA group) were separated by SDS-PAGE and visualized by western blot. The presence of either Mg++ or EDTA resulted in no difference in the molecular distribution a variety of proteins between cytosol, Triton soluble membranes, and Triton insoluble membranes. (B) Bar graphs compare the separation of membrane-associated from cytosolic proteins using high-speed centrifugation (Mg++ group) versus density gradient centrifugation (original data not shown). Black and white bars represent relative amounts of cytosolic and membrane protein (based on integrated intensity) respectively. (C) Immunoprecipitation from density gradient fractions. Fractions 5 (light membranes), 14 (dense membranes), and 17 (cytosol) were each pooled from three iodixanol gradients then split into three aliquots that contained beads coated with antibodies directed against MALS-3 (MALS-IP), control rabbit IgG, or no antibody (Start). Western blots of revealed that Dlg co-immunoprecipitated with MALS in the cytosolic and dense membrane-associated fractions. PALS1 also co-precipitated with MALS; however the PALS1 antibody detected three bands with slightly different molecular weights, and only the lower molecular weight doublet precipitated with MALS, while the higher molecular weight single band remained in the supernatant. Antibody raised against Crb3 also detected a protein that coprecipitated with MALS-3, although the molecular weight of this protein exceeded the published molecular weight of Crb3 (17 kDa) and may instead recognize Crb2 (~200 kDa). No association could be detected between MALS and β-catenin or p38γ/SAPK3. As expected, naive rabbit IgG failed to precipitate any of these proteins.

Suppl. Fig. 4

Supplementary Figure 4: Iodixanol density gradient separation of buoyant membranes from cytosol in E14 rat telencephalon homogenates. Density gradient centrifugation of homogenates from the embryonic rat telencephalon followed by a quantitative analysis of proteins present in each fraction following Western blot analysis (Vogelmann and Nelson, 2007). Briefly, CSK plus 10% iodixanol, CSK plus 20% iodixanol, and postnuclear supernatant with 30% iodixanol were overlaid and centrifuged to form a continuous density gradient. The gradient was then separated into 21 fractions from lowest to highest density, and aliquots of each fraction were run on an SDS-PAGE gel, transferred to PVDF membrane, and probed with antibodies. The integrated intensity of each band was measured, normalized to the highest value in the set, and plotted on the graph (A) (the Z-axis scale in each graph has been omitted for clarity). Protein concentration ([Protein]) of each fraction was also determined and graphed in the same manner. Particles migrated in the gradient to a position that matched their density, with cytosolic proteins remaining in the densest bottom third of the gradient (fractions 16-21) and buoyant particles in the lighter upper two-thirds of the gradient (fractions 1-15). Images of the Western blots (B) are also shown along with bars representing the distribution of cytosolic protein (actin and the majority of cellular protein, fractions 16-21), buoyant material in fractions 1 through 15, plasma membrane proteins (cadherin and the Na+/K+ ATPase, peaks at fractions 5 and 14), and membranes of the trans-golgi network (TGN38, peak centered at fraction 12). (C) Bars representing the distributions of the cytosolic, plasma membrane, and Golgi proteins in the iodixanol density gradients.

Suppl. Fig. 5

Supplementary Figure 5: MALSTKO cortices show no obvious lamination defects. E18 control and MALSTKO brains immunostained with antibodies recognizing the layer-specific markers TBR1 (layers 6-5) (A,B), CTIP2 (layer 5-6) (C,D) and SATB2 (layers 2-4) (E,F) fail to reveal obvious changes in cortical lamination. Scale bar 10 μm.

Suppl. Fig. 6

Supplementary Figure 6: Electroporation of full length MALS-3 (FL_MALS-3) does not disrupt apico-basal polarity during corticogenesis. Analyses of brains electroporated with FL_MALS-3 (A) or empty vector (B) at E13.5 reveals no change in the integrity of the VZ or the positioning of GFP+ cells at E18.5. Note that the ventricles appear normal and there are no delaminated cells present. GFP-positive cells (green) appear to have migrated normally, and immunostaining for MALS (red, A) reveals normal apical staining the VZ. (C) Breaks in β-catenin immunostaining at the apical surface of VZ is observed in brains electroporated with CA-myr_MALS3 construct. Scale bar 10μm.

Suppl. Table 1

Acknowledgements

We thank W. James Nelson and Ben Margolis for sharing their expertise and reagents, Christine Kaznowski for technical support, Yoshima Takai for MALS cDNA constructs, Thomas Sudhof for MALS-3 and Mint GST fusion constructs, Ben Margolis for MALS, PALS1 and Crb3 antibodies, Andre Le Bivic for PATJ antibodies, Sandra Martin for the pCA vector, and W. James Nelson, Jennifer Shieh, and Dino Leone for comments on the manuscript. Supported by NIH R01 MH51864 to S.K.M.

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