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Vol. 18, Issue 6, 2305-2312, June 2007
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Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1762
Submitted January 29, 2007;
Revised March 9, 2007;
Accepted March 29, 2007
Monitoring Editor: Paul Forscher
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-catenin and N-cadherin, consistent with a role in cell adhesion. Ablation of NM II-B or replacement of NM II-B with decreased amounts of a mutant (R709C), motor-impaired NM II-B in mice results in collapse of the mesh-like structure and loss of cell adhesion. This permits the underlying neuroepithelial cells to invade the spinal canal and obstruct cerebral spinal fluid flow. These defects in the CNS of NM II-B–ablated mice seem to be the cause of hydrocephalus. Interestingly, the mesh-like structure and patency of the spinal canal can be restored by increasing expression of the motor-impaired NM II-B, which also rescues hydrocephalus. However, the mutant isoform cannot completely rescue neuronal cell migration. These studies show that the scaffolding properties of NM II-B play an important role in cell adhesion, thereby preventing hydrocephalus during mouse brain development. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Crews et al., 2004).
Nonmuscle myosin (NM) II, one of the major cytoskeletal motor proteins, plays an important role in cell migration (Svitkina et al., 1997; Ma et al., 2004; Even-Ram et al., 2007; Vicente-Manzanares et al., 2007), cell–cell adhesion (Conti et al., 2004; Shewan et al., 2005; Giannone et al., 2007), and cell division (De Lozanne and Spudich, 1987; Takeda et al., 2003; Bao et al., 2005). The molecular structure of NM II is a hexamer consisting of a pair of myosin heavy chains (200 kDa) and two pairs of light chains (20 and 17 kDa). In mammals, three isoforms of the nonmuscle myosin heavy chain (NMHC) have been identified, NMHC II-A, II-B, and II-C, encoded by three different genes, Myh 9, Myh 10, and Myh 14 in humans. The NMHCs share a 60–80% identity in amino acids, but they show significant differences in their motor activities (Golomb et al., 2004; Kim et al., 2005). In general, all three isoforms are ubiquitously expressed in vertebrates with overlapping as well as different expression patterns at the cellular level. Compared with NMHC II-A and II-C, NMHC II-B is enriched in the brain, especially in neural cells. Mice ablated for NMHC II-B (B–/B–) die during the later stages of embryonic development, between embryonic day (E)14.5 and postnatal day (P)0, with abnormalities in the heart and brain (Tullio et al., 1997; Tullio et al., 2001). All B–/B– mice develop a severe, progressive hydrocephalus accompanied by disruption of the cerebral ventricular surface and distortion of the brain architecture. The causes of congenital hydrocephalus remain unknown.
In an effort to produce a mouse model of a human disease, we have generated knockin mice that carry a point mutation in the motor domain of NMHC II-B (Ma et al., 2004). We did this by mutating Arg709 to Cys, thereby duplicating a mutation that had previously been shown to occur in humans for NMHC II-A (Heath et al., 2001). Interestingly, the homologous residue has also been shown to be mutated in NMHC II-C, although, in this case, to a serine residue (Donaudy et al., 2004). In a previous report, we characterized the motor activity of a heavy meromyosin (HMM) derived from the R709C mutant myosin II-B (Kim et al., 2005). We found that, compared with wild-type HMM II-B, the mutant had only 30% of the actin-activated Mg-ATPase activity. The R709C HMM II-B also showed a very high affinity for actin and failed to propel actin filaments in the in vitro motility assay.
In generating the II-B mutant mice, we found that the presence of the cassette encoding Neomycin resistance in the mutant allele resulted in a 75% reduction in the expected mutant myosin II-B protein expression in the homozygous (BCN/BCN) mice (where C represents the R-to-C mutation, and N represents the Neomycin resistance cassette). These mice survived up to P20, and they were characterized with respect to the abnormal migration of three different groups of neurons (Ma et al., 2004). Similar to B–/B– mice, they also developed a severe hydrocephalus, but more gradually than the B–/B– mice. To understand the underlying cause of hydrocephalus in NMHC II-B–ablated and mutated mice, we made a comparative analysis of the B–/B– mice and R709C mutant mice. The purpose of the present study was to identify which properties of NM II-B are important in mouse brain development. These studies should also help to elucidate the cause of human congenital hydrocephalus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The BCN/BCN mice are maintained in a mixed background of 129/Sv and C57BL/6. To generate BC/BC mice, the floxed Neomycin resistance cassette was removed by crossing the B+/BCN mice with BALB/C CMV-Cre mice (The Jackson Laboratory, Bar Harbor, ME). All procedures were conducted using an approved animal protocol in accordance with the National Heart, Lung, and Blood Institute Animal Care and Use Committee.
Histology and Immunofluorescence Staining
The embryos were collected in phosphate-buffered saline (PBS) and directly immersed in 4% paraformaldehyde in PBS, pH 7.4, overnight. Paraffin sections at a thickness of 5 µm were prepared by Histoserv (Germantown, MD) for hematoxylin and eosin (H&E) or immunofluorescence staining. The following primary antibodies were used in this study: polyclonal antibodies against NMHC II-A (1:1000), II-B (1:3000), and II-C (1:1000; Phillips et al., 1995; Buxton et al., 2003); monoclonal antibodies against N-cadherin (1:200; Zymed Laboratories, South San Francisco, CA), -catenin (1:200; Zymed Laboratories), serine-19 phospho-regulatory myosin light chain (pMLC20) (1:100; Cell Signaling Technology, Danvers, MA), atypical protein kinase C (aPKC)
(1:100; BD Biosciences, San Diego, CA), and myosin light chain kinase (1:1000; Sigma-Aldrich, St. Louis, MO). Fluorescence secondary antibodies used were Alexa 488 goat anti-rabbit immunoglobulin G (IgG) or Alexa 594 goat anti-mouse IgG (1:250; Invitrogen, Carlsbad, CA). The images were collected using an SP Confocal microscope (Leica, Wetzlar, Germany). In all cases, when possible, comparison was made among littermates. For each genotype in each experiment, we analyzed at least five mice.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Ma et al., 2004). However, the BCN/BCN mice develop hydrocephalus more gradually than the B–/B– mice, and with less destruction and distortion of the ventricles and the surrounding brain tissue. Figure 1 shows sagittal and coronal sections of BCN/BCN mouse brains showing evidence of mild dilatation of the cerebral ventricles at E14.5 (Figure 1b) and increased dilatation of the cerebral ventricles as well as the aqueduct of Sylvius at P0 (Figure 1, d and f). As reported previously, B–/B– mice show marked enlargement of the cerebral ventricles as early as E12.5 and a severe disruption and distortion of the ventricles by P0 (see figure 9 in Tullio et al., 2001).
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-catenin. It is important to note that, during this period of brain and spinal cord development, many of the neuroepithelial cells extend processes that reach the ventricular border and that the mesh-like structure we describe is present at the apical border of all these cells.
Figure 6, a–c, shows a magnified view of the mesh-like structure bordering the spinal canal immunostained for NM II-B (green), -catenin (red) at E11.5 in a wild-type mouse. The importance of NM II-B to the structure of this complex is shown by the collapse of the mesh-like configuration in B–/B– mice at E11.5 (Figure 6, compare h and i with g). In BCN/BCN mice, the structure begins to fall apart at E11.5 (Figure 6f), and it is almost gone by E12.5 (Figure 6e). Figure 6f (arrow) shows an early example of the protrusion of a neuroepithelial cell into the spinal canal of a BCN/BCN mouse. This suggests that cell–cell adhesion has already become weakened in this area, although the general configuration still remains. Figure 6h shows that loss of NMHC II-B not only results in the collapse of the mesh-like structure at the apical border but also results in discontinuity of the apical structure lining the spinal canal. The disruption of the structure permits the underlying neuroepithelial cells to invade the spinal canal and obstruct the flow of CSF.
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). Interestingly, increasing the expression of R709C mutant NMHC II-B in BC/BC mice restored an intact spinal cord ventricular surface. Figure 7b shows that the spinal canal of BC/BC mice is free of obstruction, and Figure 7, d, f, and h, shows that the mesh-like structure present at the apical border of the cells lining the spinal canal has been restored. This finding suggests that the loss of the mesh-like structure and decrease in cell adhesion were not solely due to the mutation in NMHC II-B, but due to the 75% decrease in NMHC II-B. Importantly, no evidence for hydrocephalus was observed in BC/BC mice as late as E16.5, the time when they succumbed to cardiac defects. These results further support the idea that obstruction of the spinal canal seen in B–/B– and BCN/BCN mice during embryonic development causes the development of the hydrocephalus.
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). This suggests that the restoration of the integrity of the neuroepithelial cells lining the spinal canal by increasing the amount of mutant NM II-B relies more on the structural properties of NM II-B than on its motor activity. Because the motor activity of NM II is regulated by phosphorylation of the 20-kDa regulatory myosin light chain (MLC20), it was of interest to examine the state of light chain phosphorylation in B+/B+ and BC/BC mice in the cells bordering the spinal canal. No evidence for MLC20 serine-19 phosphorylation could be detected in these cells by using MLC20 serine-19 phospho-specific antibodies (Figure 8a), although there is evidence for serine-19 phosphorylation in the nearby vasculature (arrows). These findings support the idea that, although NM II-B is required to maintain cell–cell adhesion at the apical border of the spinal canal, activation of its motor is not required.
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showed that conditional ablation of aPKC in developing mouse brains resulted in the loss of adhesion junctions in the neuroepithelial cells lining the cerebral ventricles. We, therefore, used antibodies to aPKC to analyze for the presence of this kinase in the spinal cord at E12.5. As shown in Figure 8c, aPKC is enriched at the apical border of the neuroepithelial cells lining the spinal cord similar to NMHC II-B (Figure 8d). A similar result was obtained at E11.5. Conversely, myosin light chain kinase is not present in these cells, although it is expressed in the nearby vasculature (Figure 8b, arrows).
Mutant Myosin II Fails to Fully Rescue Impaired Neuronal Migration
Expression of wild-type quantities of R709C mutant NM II-B in BC/BC mice rescued the cell–cell adhesion defect and hydrocephalus seen in B–/B– and BCN/BCN mice. We next examined whether other brain defects found in the BCN/BCN mice, such as the impaired migration of facial and pontine neurons, were also rescued by increased expression of the mutant NM II-B in BC/BC mice. Because BC/BC mice die by E16.5 from cardiac defects, we are unable to look at cerebellar granule cell migration, which is also impaired in the hypomorphic BCN/BCN mice (Ma et al., 2004). Figure 9 shows H&E-stained sagittal sections of the developing hindbrains from wild-type and BC/BC mice at E16.5. The figure shows pontine neurons in BC/BC mice that were arrested in their migration. The direction of migration is from left to right (arrows in Figure 9, a and c), and many of the BC/BC neurons are delayed in their migration as indicated by their location in the lower left of the lateral sagittal section (Figure 9c). Figure 9, b and d, shows medial sagittal sections in which many of the pontine neurons of B+/B+ mice have reached their final destination by E16.5, whereas none of the pontine neurons have reached their final destination in BC/BC mice (compare arrows in Figure 9, b and d; n = 10 for each). In the case of facial neuron migration, 20% of the BC/BC mice showed an impaired migration similar to that shown for BCN/BCN mice (Figure 1b, arrow).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell–Cell Adhesion of the Neuroepithelial Cells Requires Nonmuscle Myosin II, but Not Activation of Its Motor Domain
Our finding of a transitory mesh-like structure containing a myosin complex is supportive of a structural role for NM II, and it is consistent with our ability to rescue hydrocephalus by using a motor impaired myosin. Because NMHC II-B is the only isoform present in the neuroepithelial cells lining the spinal canal, ablation of this isoform renders these cells more vulnerable to abnormalities than other cells, such as the epithelial cells lining the airways and intestines where all three isoforms of nonmuscle myosin II are expressed (Golomb et al., 2004). The finding of a unique mesh-like adhesion structure that includes NM II-B along with N-cadherin and -catenin prompted us to postulate that NM II-B is involved in maintaining cell adhesion at the apical border of the neuroepithelial cells lining the spinal canal. As expected, we also detected actin in the mesh-like structure and the actin pattern was similar in both B+/B+ and BC/BC mice (unpublished data). Involvement of NMHC II-B in cell adhesion among these cells was also reported by Hildebrand (2005).
To define which properties of the NMHC II play a role in vivo in maintaining cell adhesion in these cells, we made use of a mutated form of NM II-B (R709C), which has been shown to bind to actin with high affinity, but cannot propel actin filaments in the in vitro motility assay (Kim et al., 2005). Replacement of NM II-B with wild-type amounts of mutant myosin restored normal cell adhesion and prevented hydrocephalus. The observation that increasing the quantity of a mutant form of myosin II can rescue cell adhesion is consistent with the idea that the scaffolding properties of myosin are of greater importance than its motor activity in these circumstances. Previous work by others supports the concept of the importance of the structural properties of myosin II in Dictyostelium (Xu et al., 2001). Further evidence of a structural role for myosin in the apical complex is supported by our inability to detect significant activation of NM II-B, as reflected by the lack of serine-19 phosphorylation of MLC20 at the apical border of the neuroepithelial cells. Furthermore, activation of myosin is not required for maintaining cell adhesion, and it has been reported to disrupt the adhesion junctions in cancer cell lines (Sahai and Marshall, 2002).
Of note are our recent findings that NM II-A can also rescue hydrocephalus and the defects in cell adhesion in the spinal cord of B–/B– mice when it replaces endogenous NM II-B in the neuroepithelial cells of the spinal cord (unpublished data). Rescuing these defects in B–/B– mice with NM II-A further supports the idea that maintaining cell–cell adhesion in the spinal neuroepithelial cells is not dependent on an isoform-specific function of NMHC II-B, but on a function shared by both II-A and II-B. Although both NM II-A and II-B show a high affinity for actin, there are significant differences in their kinetic motor properties (Kovacs et al., 2003). For example, NM II-B has a duty ratio (portion of the kinetic cycle spent in a state strongly bound to actin) that is more than an order of magnitude higher than that of NM II-A. This is consistent with our proposal that a structural property of NM II and not its motor activity is required to maintain cell–cell adhesion in the neuroepithelial cells.
In addition, we show here that aPKC is also enriched in this adhesion structure together with NMHC II-B. Although it is possible that NMHC II-B serves as a downstream target of aPKC in regulating cell adhesion in the spinal canal, there are other putative targets in the neuronal adherens junction as well, such as PAR3 and Lgl1 (Yamanaka et al., 2003; Klezovitch et al., 2004; Suzuki and Ohno, 2006; Vasioukhin, 2006). In contrast, Even-Faitelson and Ravid (2006) have reported that phosphorylation of the NMHC II-B in the nonhelical tail by an aPKC results in cortical localization of both NM II-B and aPKC in a prostate cancer cell line. The requirement of aPKC in cell–cell adhesion has been demonstrated both in cultured cells (Suzuki et al., 2001, Nunbhakdi-Craig et al., 2002) and neuroepithelial cells in the developing mouse brain (Manabe et al., 2002). As noted above, the importance of aPKC in maintaining adherens junctions in the neocortex was demonstrated by conditional loss of this enzyme, which also resulted in hydrocephalus, although at a much later time (P3) than we report here (Imai et al., 2006). This report on the role of the scaffolding properties of NM II raises important and intriguing questions. How does a nonmotoring NM II contribute to the generation of an adhesion structure? What is the exact relationship between NM II and the cell adhesion complex? These questions will be the subject of future experiments both in vitro and in vivo.
Migration of Pontine Neurons Requires NMHC II-B–specific Motor Activity
Although expression of wild-type quantities of the motor-impaired R709C mutant NM II-B in BC/BC mice rescued cell adhesion in the spinal cord, abnormalities in migration of the pontine neurons and facial neurons persisted. Similarly, replacement of NM II-B by II-A also failed to rescue the defects in neuronal migration. Unlike the properties of myosin that are responsible for cell adhesion, migration of these neuronal cells is more likely dependent on the motor function of myosin II-B. This is also consistent with our finding that MLC20 of NM II-B is phosphorylated in the migrating pontine neurons during brain development (Ma et al., 2004).
In summary, our studies show that NM II-B plays an important role in cell adhesion at the apical border of the cells lining the spinal canal during mouse brain development. The cell adhesive properties of the neuroepithelial cells lining the canal depend more on the structural properties of NM II-B than on its motor activity. These findings also shed light on the role of NM II-B in the pathogenesis of congenital hydrocephalus.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Xuefei Ma (max{at}nhlbi.nih.gov)
Abbreviations used: aPKC, atypical protein kinase C; CSF, cerebral spinal fluid; E, embryonic day; HMM, heavy meromyosin; MLC20, regulatory myosin light chain; NM, nonmuscle myosin; NMHC, nonmuscle myosin heavy chain; P, postnatal day.
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