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Vol. 19, Issue 5, 2289-2299, May 2008
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*Electron Microscope Laboratory and Organogenesis and Neurogenesis Group, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan
Submitted December 26, 2007;
Revised February 19, 2008;
Accepted February 29, 2008
Monitoring Editor: Yu-Li Wang
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Jaffe and Hall, 2005). The GTP-bound active form of Rho-GTPases can interact with their effector proteins and thus control various aspects of cell behavior. To date, numerous studies have examined the multiple signal transduction pathways of Rho-GTPases in extensive detail at the biochemical and cellular levels, but little is known yet about how these proteins work in vivo in vertebrate morphogenesis. Several lines of Rho-GTPase knockout mice showed early embryonic lethality (Sugihara et al., 1998; Chen et al., 2000), indicating an essential role for such regulators, although the results also suggested that a genetic approach is not ideal for analysis of the functions of these proteins throughout development. In addition, the precise endogenous distribution of Rho-GTPases has not been investigated because of the lack of reliable antibodies for staining. We have been successful recently in developing specific antibodies and suitable staining conditions for visualizing the distribution of Rho (Yonemura et al., 2004). Here we report that Rho accumulates in the apical region of neural plate cells during neural tube formation.
The neural plate—the thickened ectoderm—bends and fuses to form the neural tube through a series of precisely orchestrated morphogenetic movements (see Figure 1A; Smith and Schoenwolf, 1997). Rearrangement of neural plate cells drives mediolateral narrowing and rostrocaudal elongation of the neural plate. Further mediolateral narrowing and bending (or rolling) of the neural plate result from changes in the shape of the neural plate cells, especially by apical constriction. Because the driving forces are generated by the coordinated remodeling of cell shape and adhesion, cytoskeletal reorganization and regulation are essential to neurulation. Several lines of knockout mice lacking cytoskeleton-associated proteins, such as p190 RhoGAP, shroom, Mena, MARCKS, and vinculin, have shown defects in neurulation (Wu et al., 1996; Xu et al., 1998; Hildebrand and Soriano, 1999; Lanier et al., 1999; Brouns et al., 2000). Exactly how these proteins spatiotemporally regulate the cytoskeleton and how such activities are regulated during neurulation remain elusive.
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). One of the members of the shroom family, shroom3, is associated with both the actomyosin cytoskeleton and apical constriction of neural plate cells (Hildebrand and Soriano, 1999; Haigo et al., 2003; Hildebrand, 2005), but is regulated by Rap1, not Rho (Haigo et al., 2003). Seen in this light, therefore, the molecular mechanisms of cytoskeletal reorganization during neurulation and the involvement of Rho remain issues for further exploration.
Rho-GTPases are regulated by several morphogenetic signals, such as Wnt, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF; Habas et al., 2003; Harada et al., 2005; Theriault et al., 2007; Zhang et al., 2007), among which the Wnt/planar cell polarity (PCP) signaling pathway is the most studied (Habas et al., 2003; Veeman et al., 2003). Wnt signals include the canonical signaling pathway that leads to β-catenin–mediated transcriptional regulation and several noncanonical signaling pathways (Kikuchi et al., 2006). One noncanonical pathway, the Wnt/PCP pathway, is an important element of cell rearrangement within single-cell layers. In Xenopus gastrulation, it regulates convergent extension movement (Tada et al., 2002), and activation of the Rho-GTPases downstream of the Wnt/PCP signal is required for this morphogenesis (Habas et al., 2001, 2003). Recent studies indicate that the Wnt/PCP signal during neurulation is an important element of neural plate cell rearrangement (Wallingford and Harland, 2002; Ciruna et al., 2006; Wang et al., 2006; Ybot-Gonzalez et al., 2007), although further investigation is required to explore the involvement of Rho-GTPases downstream of the Wnt/PCP signal in this process.
In this study we report on the coordination between Rho activity, myosin activity, and neural plate dynamics and the need for Rho and Rho-kinase activity for correct neurulation. In addition, activation of Rho is required for its own apical accumulation in neural plate cells. Inhibition of the Wnt/PCP pathway causes disruption of the apical accumulation of Rho in neural plate cells, abnormal morphology of neural plate cells, and the failure of neural tube development. Our data, based on endogenous protein distribution, delineate the physiological function and importance of Rho in the morphogenetic movement of the neural plate and further our understanding of the molecular mechanisms of cytoskeletal reorganization in neural plate cells during neurulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), was kindly donated by Dr. S. Narumiya (Kyoto University, Japan). pGEX-4T-1-N-Tat-C3 was constructed as follows. Tat-tag is derived from human immunodeficiency virus and is a cell-penetrating peptide tag (Suzuki et al., 2002). A DNA fragment containing the region coding Tat-tag (oligonucleotides 5'-GATCAGGCAGGAAGAAGCGGAGACAGCGACGAAGACCTCCTCAAGGATCCCCGG-3' and 5'-AATTCCGGGGATCCTTGAGGAGGTCTTCGTCGCTGTCTCCGCTTCTTCCTGCCT-3' were annealed) was inserted into the BamHI-EcoRI site of pGEX-4T-1 (GE Healthcare, Little Chalfont, United Kingdom) to construct pGEX-4T-1-N-Tat. This has the same multicloning site as pGEX-4T-1, and Tat-tag is located just after the thrombin digestion site and immediately before the multicloning site. The sequence encoding botulinum toxin C3 (Kumagai et al., 1993) from the vector pEGFP-C3 (donated by Dr. S. Narumiya) was subcloned into the BamHI site of pGEX-4T-1-N-Tat. The chick expression vector pCA MCS (–) (Niwa et al., 1991) was kindly donated by Dr. H. Niwa (RIKEN CDB, Japan) and was amino-terminally tagged with enhanced green fluorescent protein (EGFP) to generate pCA-EGFP-C1 by inserting the NheI-BamHI–digested EGFP-containing fragment from pEGFP-C1 (Clontech, Mountain View, CA) into the NheI-BglII site of pCA MCS (–). pEF-BOS-Myc-RhoA, carrying a constitutively active Rho mutant (Rho-CA, V14G), was kindly donated by Dr. K. Kaibuchi (Nagoya University, Japan). Rho-CA and C3 were subcloned into the BglII site of pCA-EGFP-C1. For Xenopus microinjection experiments, we used the mRNA production vectors pCS2-Venus (a GFP mutant kindly donated by Dr. A. Miyawaki, RIKEN BSI, Japan; Nagai et al., 2002), pCS2-Xdsh (Xenopus dishevelled wild type), pCS2-Xdd1 (Xenopus dishevelled dominant negative form; Sokol, 1996), pCS2-XWnt11-dC (Xenopus carboxy-terminal region lacking Wnt11 dominant negative form; Tada and Smith, 2000), and pCS2-XFrz7-dC (Xenopus carboxy-terminal region lacking Frizzled7 dominant negative form; Djiane et al., 2000).
Antibodies
Anti-Rho rat mAb was raised and characterized as described previously (Yonemura et al., 2004). Anti-chick β-catenin rabbit serum was kindly donated by Dr. M. Takeichi (RIKEN CDB, Japan). Anti-myosin light chain 2 (MLC2) and anti-phospho-MLC2 (Thr18, Ser19) rabbit polyclonal antibodies were purchased from Cell Signaling (Danvers, MA). Anti-GFP rabbit serum and Alexa-Fluor 488-, 647-phalloidin were purchased from Invitrogen (Tokyo, Japan).
Immunocytochemistry
Chick and Xenopus embryos cultured by conventional methods were fixed with 4% paraformaldehyde in 0.1 M HEPES buffer (pH 7.5) or 10% trichloroacetic acid (TCA) for 2 h at 4°C. For cryoprotection, specimens were placed into 0.1 M HEPES buffer (pH 7.5) containing 30% sucrose for 12 h at 4°C. The specimens were frozen in OCT compound (Sakura, Tokyo, Japan) and cut into sections (16 µm thickness) by cryostat. The sections were permeabilized with phosphate-buffered saline (PBS; pH 7.2) containing 30 mM glycine (G-PBS) and 0.2% Triton X-100 for 5 min and blocked with G-PBS containing 4% normal donkey serum for 30 min. The sections were incubated with the primary antibody for 1 h (anti-Rho, 1:200; anti-β-catenin 1:200, anti-phopsho-MLC2 1:100, and anti-GFP 1:500), washed, and incubated with secondary antibodies for 30 min. All antibodies were diluted with G-PBS containing 4% normal donkey serum. Specimens were observed through a confocal microscope (LSM 510, Carl Zeiss, Tokyo, Japan).
Pulldown Assay of GTP-bound Rho-GTPases
Neural plate regions were cut from chick embryos with fine tungsten needles and immediately frozen in liquid nitrogen. In the case of Hamburger and Hamilton stage 8–9 (HH 8–9) embryos, only neural plate regions (unclosed neuroepithelial regions) were isolated, whereas in the case of HH 12–13 embryos, only neural tube regions (closed neuroepithelial regions) were isolated. For each assay, 40–50 HH 5–6 embryos, 
25 HH 8–9 embryos, and 20 HH 12–13 embryos were collected. The G-LISA RhoA Activation Assay Biochemistry Kit (Cytoskeleton, Denver, CO) was used for the detection of Rho activity. For immunoblotting data, however, we performed the assay with glutathione S-transferase (GST)-RBD protein purified from Escherichia coli (80 µg of GST-RBD protein and 20 µl [bed volume] of glutathione Sepharose 4B [GE Healthcare] per experimental point). All procedures and buffers were the same as those for the G-LISA Kit, except that bound proteins were eluted with 100 µl of Laemmli buffer and were detected by immunoblotting.
Detection of Phosphorylation of MLC
Chick embryos were fixed in 10% TCA for 1 h at 4°C. This treatment allowed specific neuroepithelial cells to be isolated easily with forceps. In the case of HH 8–9 embryos, only neural plate regions were isolated, whereas for HH 12–13 embryos, only neural tube regions were isolated, as above. For each assay, 25 HH 5–6 embryos, 15 HH 8–9 embryos, and 12 HH 12–13 embryos were collected. The samples subsequently underwent immunoblot analysis with anti-MLC and anti-phospho-MLC.
Inhibitor Experiments with Chick Embryos
Chick embryos were cultured on albumen-agar plates as described previously (Sundin and Eichele, 1992), but with slight modifications. Inhibitors (cytochalasin D [CD], Y27632, and (–) blebbistatin; Merck Calbiochem, Tokyo, Japan) were diluted with Hanks' balanced salt solution (Invitrogen) containing 0.01% saponin instead of with Yolk-Tyrode medium. Inhibitor solutions were added individually to albumen-agar plates (0.5 ml per 6-cm dish) 30 min before use. A filter paper circle carrying an HH 5–6 embryo dorsal side up was placed on the agar plate and overlaid with 20 µl of inhibitor solution. The vitelline membrane was then broken with forceps to expose the embryo to the inhibitor solution. The embryo was preincubated at room temperature for 2 h to enable the inhibitor to penetrate while development was suspended. Excess inhibitor solution was then removed, 20 µl of fresh inhibitor solution was added carefully, and the plate was incubated at 38.5°C (time 0) to resume embryonic development. Every 3 h, the inhibitor solution was replaced. After 8–9 h of incubation, when the development of control embryos had reached about HH 9, the samples were fixed. Purified GST-Tat and GST-Tat-C3 were obtained from a bacterial protein expression system, GST Gene Fusion System, according to the manufacturer's instructions (GE Healthcare), and Tat-C3 was obtained by using thrombin to remove GST.
Electroporation of Chick Embryos
HH 5–6 embryos were electroporated as described previously (Kobayashi et al., 2002), but at 5 V, on a model CUY21 EDIT electroporator (NEPA GENE, Ichikawa, Japan).
Microinjection of Xenopus Embryos
Embryos were microinjected with mRNA as described previously (Mizuseki et al., 1998). Embryos were fixed in 10% TCA (4°C, 2 h) at neurula stage.
Scanning Electron Microscopy
Neurula Xenopus embryos were fixed in 1% glutaraldehyde in PBS (4°C, 8 h). They were postfixed in 0.5% osmium oxide for 90 min at 4°C, dehydrated through a graded ethanol series, and transferred into isopentyl acetate. The specimens were then critical-point dried, platinum coated, and analyzed under scanning electron microscope (JSM-5610LV, JEOL, Tokyo, Japan).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Interestingly, we also found that after neural tube closure, the apical accumulation was reduced (Figure 1Bb). These results suggest that the Rho accumulation in the apical region of the neural plate coincides with morphogenetic activity.
Rho Localizes at Active Actomyosin-enriched Sites
The apical constriction of neural plate cells is thought to be based on an actomyosin network associated with junctional complexes. Rho has been implicated in the formation of adherens junctions and in actomyosin contraction through phosphorylation of MLC (Jaffe and Hall, 2005). Accumulated Rho likely reorganizes or maintains adherens junctions and their associated actomyosin bundles in neural plate cells that exert apical constriction force. The distribution of Rho partly overlapped with actin filaments in the apical region of neural plate cells (Figure 2A), and Rho further accumulated to spot-like sites largely merged with β-catenin, a known constituent of adherens junctions (Figure 2B). The colocalization of Rho and phosphorylated MLC (Figure 2C) indicates that Rho accumulated in sites where myosin II was activated.
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Activities of Rho and Myosin II in the Neural Plate Are Related to the Dynamics of Neurulation
Activated Rho-GTPases localize at plasma membranes (Takai et al., 1995; Van Aelst and D'Souza-Schorey, 1997), and the Rho that accumulated apically certainly seemed to be active. To confirm this speculation, we collected neural plates dissected from embryos and biochemically measured the activity of Rho in the neural plate. The activity of Rho was highest at HH 8–9 (Figure 4A), in agreement with the histochemical data of Rho accumulation. We next examined myosin II activity in the neural plate by measuring the amount of phosphorylated MLC. Like Rho, the activity of myosin II in the neural plates was highest at HH 8–9 (Figure 4B). Because the morphogenetic movement of primary neurulation is most dynamic at HH 8–9, the peak activities of Rho and myosin II are coincident with the morphogenetic activity of neurulation.
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; Park et al., 2003); however, in many cases, the concentration of inhibitor was extremely high compared with concentrations often used in cell biology, perhaps because inhibitors cannot penetrate into embryos as easily as cultured cells. Because we cannot exclude the possibility that high concentrations of inhibitors might lead to undesirable side effects, we developed a more effective drug delivery protocol for use in chick embryos. Some specific aspects of this improved system include direct exposure of the dorsal surface of embryos to inhibitor solutions by cutting the vitelline membrane, addition of a mild detergent in inhibitor solutions to increase penetration, and preincubation of embryos in inhibitor solutions at room temperature before resumption of development by shifting the incubation temperature to 38.5°C. In the vast majority of cases (88%), control embryos treated according to this protocol but in the absence of inhibitors developed normally (Table 1).
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), neurulation of the chick embryo is affected by CD, which inhibits actin polymerization (Figure 5B and Table 1). In most cases, exposure to 1 µM CD caused severe defects, including nearly halting neurulation and thinning and flattening of the neural plate (Figure 5B, c and d). These results indicate that the actin cytoskeleton is necessary to maintain the columnar shape of neural plate cells. Several embryos showed mild defects (mainly in experiments using 0.2 µM CD), successfully completing neural tube closure at the midbrain level but failing to complete closure at the forebrain and spinal cord levels (Figure 5Ba). These mild phenotypes are the same as those described in the previous report (Schoenwolf et al., 1988), even though the concentration of CD used in the previous study was much higher (1.6 µg/ml 
3.2 µM).
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C3 is a component of a botulinum toxin and an inhibitor of Rho. Tat-tag is a penetrating peptide tag; Tat-C3 has proved to be an effective tool for inhibiting Rho (Park et al., 2003). Exposure to Tat-C3 caused a severe phenotype (Figure 5D and Table 1), and the apical accumulation of Rho was decreased dramatically (Figure 5Dg), indicating that activation of Rho is necessary for its localization in the apical region. Exposure to C3 also inhibited MLC phosphorylation (Figure 5Dh). These results suggest that active Rho localizes in the apical region of the neural plate and generates myosin-mediated apical constriction, which is necessary for correct neurulation.
Y27632 inhibits Rho-kinase, a key effector of Rho and regulator of MLC phosphorylation (Zhao and Manser, 2005). Although a previous study cursorily assessed the effect of 200 µM Y27632 on neurulation (Wei et al., 2001), the high concentration used might have inhibited several other kinases in addition to Rho-kinase. In the current study, we succeeded in reducing the concentration to levels similar to those used in cell biology (50 µM). Exposure to Y27632 caused failure of neural tube closure or resulted in an abnormal neural plate, and we saw wavy neural plates (Figure 5E and Table 1). These phenotypes were all very similar to those produced by treatment with blebbistatin. Subsequent immunoblots showed a Y27632-induced reduction in MLC phosphorylation (Figure 5Ed). These data suggest that the Rho–Rho-kinase pathway plays an important role in correct neurulation via regulation of myosin II activity.
Correct Regulation of Rho Activity Is Important for Neurulation
Although it is reasonable to assume that actomyosin constriction is essential for correct neurulation, the importance of the activation of Rho has remained unclear because the p190 RhoGAP knockout mouse showed neurulation defects (Brouns et al., 2000). We observed the effects of overstimulation of Rho on neurulation (Figure 6). Overstimulation of Rho by expression of its constitutively active mutant (Rho-CA) caused rounding of cells, some of which were extruded from the neural tube into the apical lumen (Figure 6, C and D), whereas neural plate cells expressing only GFP after electroporation were columnar in shape, just as were normal neural plate cells (Figure 6, A and B). Inhibition of Rho due to expression of C3 also caused rounding of cells and extrusion from the neural tube to the apical lumen (Figure 6, E and F). At sites in the neural tube that contained many Rho-CA- or C3-expressing cells, the neural tube was thin or disordered (Figure 6, D and F). These results suggest that not only activation of Rho, but also its inactivation, is important for correct neural tube cell shape and neural tube formation.
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). The Wnt/PCP signal is also important for proper neurulation throughout vertebrates (Wallingford and Harland, 2002; Ciruna et al., 2006; Wang et al., 2006; Ybot-Gonzalez et al., 2007). Therefore, it is reasonable to think that activation and accumulation of Rho in the neural plate are regulated by the Wnt/PCP pathway. Mutant molecules that affect the Wnt signal transduction pathway have been established in the Xenopus system, and we detected apical accumulation of Rho in the Xenopus neural plate (Figure 7C, a–c). We therefore decided to use Xenopus embryos in an attempt to explore the relationship between the Wnt/PCP signal and the accumulation of Rho.
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). The expression of Xdd1 caused neurulation defects with broad neural plates, as previously reported (Figure 7B; Wallingford and Harland, 2002). These broad neural plates had a reduced apical accumulation of Rho (Figure 7C, d–f), and these Xdd1 phenotypes were partially rescued by coexpression of wild-type Xdsh (Figure 7C, g–j). These results implicate Xdsh activity in the apical accumulation of Rho in the Xenopus neural plate, probably through Rho activation.
To further confirm the relationship between the Wnt/PCP pathway and apical accumulation of Rho, we inhibited the Wnt/PCP pathway by using other specific molecules. A Wnt receptor, Xenopus frizzled7 (XFrz7), and one of its ligands, Xenopus Wnt11 (XWnt11), both specifically stimulate the Wnt/PCP pathway (Tada et al., 2002), and the C-terminal deletion mutants of these proteins (XFrz7-dC and XWnt11-dC) are their dominant negative forms (Djiane et al., 2000; Tada and Smith, 2000). Like Xdd1, expression of XFrz7-dC and XWnt11-dC affected neurulation and decreased the apical accumulation of Rho (Figure 7D).
The Wnt/PCP pathway regulates cell rearrangement in the neural plate in the same way as it regulates convergent extension in gastrulation, because inhibition of the Wnt/PCP pathway causes the neural plate to have a smaller length-to-width ratio than that of wild-type embryos, with no effect on tissue differentiation (Wallingford and Harland, 2002; Wang et al., 2006; Ybot-Gonzalez et al., 2007). However, little is known about cell shape in the neural plate in Wnt/PCP-inhibited embryos. We used scanning electron microscopy to assess the cell surface of the neural plate. Normal neural plate cells were arranged tightly and densely in the neural groove, and the apical cell surface was long and narrow along the anterior–posterior axis. In contrast, Xdd1-injected neural plate cells were arranged irregularly (Figure 7E), they were wider than normal, and their area was increased. This observation suggests that the smaller length-to-width ratio of the neural plate in Wnt/PCP-inhibited embryos results not only from inhibition of cell rearrangement, but also from the smaller length-to-width ratio and the increased surface area of each neural plate cell. These changes in cell shape are likely caused by inhibition of actomyosin apical constriction downstream of the Wnt/PCP-Rho pathway.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Bertet et al., 2004); and Rho, Rac, and Cdc42 regulate cytoskeletal reorganization of dorsal closure (Harden et al., 1999). In mice, the Rho–Rho-kinase pathway is involved in eyelid formation (Shimizu et al., 2005). Many studies of the function of Rho-GTPases in vertebrate morphogenesis focus on Xenopus gastrulation, in which the Wnt/PCP pathway leads to cell rearrangement through Rho activation (Tada et al., 2002; Veeman et al., 2003). Genetic approaches like those just described are not always sufficient for analysis of the spatiotemporal-specific function of Rho-GTPases, because the phenotypes are the sum of all effects during development. In addition, the fundamental nature of Rho-GTPases in so many cellular processes means that they are likely to be implicated throughout morphogenesis, and the morphogenetic event that most depends on Rho-GTPase remains undetermined. In the current study, we show the physiological importance of Rho in neurulation, based on our observations of when, where, and to what extent Rho accumulates.
We found that Rho accumulates predominantly near the apical region of bending neural plates. Its overlapping localization with actin filaments, β-catenin, and phosphorylated MLC supports the idea that its accumulation causes apical constriction and concomitant cytoskeletal reorganization in the neural plate. In the anterior dorsolateral region, the accumulation of Rho was maintained during second bending, but decreased after neural tube closure. This spatiotemporal-specific accumulation suggests the functional significance of Rho in the morphogenesis of neurulation. Further, the apical accumulation of Rho at the first bending point was less than and in a narrower region than that seen at the second bending. This pattern suggests that the lower constriction force produced by a smaller number of cells is sufficient for the first bending, probably because forces from outside the neural plate (extrinsic forces) are dominant for this bending, indicating that the first bending of the neural plate may be a rather passive movement. Extrinsic forces are caused by the proliferation and expansion of epidermal ectodermal cells adjacent to the neural plate. In the posterior part of the bending neural plate, Rho accumulated throughout the apical region of dorsolateral cells, which have no obvious hinge point. The posterior spinal cord neural tube has a much smaller diameter than has the anterior brain neural tube, whereas the thicknesses of both the anterior and posterior neural plates are similar. Curving of a layer of the same thickness with a smaller radius of curvature requires a greater apical constriction force. Even though the dorsolateral regions of the posterior neural plate lack an obvious hinge point, apical constriction at levels similar to those during the second bending of the anterior neural plate may be required on account of the decreased radius posteriorly.
In this study we demonstrate that the high levels of Rho activity as measured by biochemical assays are related to dynamic movement during neurulation. We have also been able to use specific inhibitors of actin, myosin II motor, Rho, and Rho-kinase during neurulation to show that each caused various defects in neural tube closure. In addition, we showed that the activation of Rho is necessary for its own apical localization in neural plate cells and confirmed that inhibition of Rho or Rho-kinase leads to a biochemical reduction in myosin II activity. Together our findings indicate that the active form of Rho that accumulates near the apical region regulates the cytoskeletal reorganization required for correct neurulation. Inhibition of Rho kinase or myosin II caused wavy neural plates; this wavy phenotype is indicative of a loose neural plate, which is probably due to failure of apical surface constriction and cellular rearrangement. Because the second bending does not occur in the posterior part of the neural plate (Figure 1A), the 
-like structures seen in Figure 5, Cc and Ec, did not result from normal bending, but suggest that both the right and left side of the loose neural plate were pushed upward by extrinsic forces. Our observations also suggest that the myosin II and Rho kinase activity is important in generating intrinsic forces in the neural plate, but is less so for extrinsic forces.
In addition to the transient accumulation and activation of Rho at the neural plate cells during neural tube formation and the requirement of Rho activity for neural tube formation, we showed that continuous activation or inhibition of Rho in neural plate cells by using RhoA-CA or C3, respectively, results in defects in neurulation (Figure 6). These results indicate that not only Rho activity, but also its correct spatiotemporal regulation, is required for neural tube formation.
shroom3 is one of the most important actin-associated proteins involved in the apical constriction of neural plate cells (Hildebrand and Soriano, 1999; Haigo et al., 2003; Hildebrand, 2005), and one study has reported that shroom3 is regulated by Rap1 but not by Rho (Haigo et al., 2003). However, because both Rho and shroom3 coordinately contribute to apical constriction of epithelial cells (Hildebrand, 2005), they might also work together in different pathways in neurulation. Because the functional link between shroom3 and Rap1 was examined only in blastula epithelial cells in the cited study, the possibility remains that shroom3 is regulated by Rho in neuroepithelial cells at the neurula stage. Further study is needed to explore the regulatory pathway of shroom3-mediated apical constriction of neural plate cells.
The most-studied morphogenetic signaling pathway underlying Rho activation is the Wnt/PCP pathway, which also is involved in neurulation. However, the link between Rho activation and neurulation has remained obscure. Although some reports suggest the involvement of Rho-kinase in neural tube formation (Wei et al., 2001; Ybot-Gonzalez et al., 2007), the changes in Rho activity and its downstream target activity in neural plate regions had not been measured directly. In the current study, we used biochemical means to quantify the temporal activation of Rho and Rho-kinase in neural plate regions and found that Rho activity is essential for its apical accumulation. Considering that inhibition of the Wnt/PCP pathway disrupted the apical accumulation of Rho and apical constriction, this pathway likely regulates Rho activity in the neural plate during neurulation.
PCP causes orientated cell migration and changes in cell shape within cell layers and often directs the morphogenetic movement of epithelium-like cell layers. The noncanonical Wnt signaling pathway regulates this morphogenetic movement through several developmental events (Veeman et al., 2003). During neurulation, PCP leads to rearrangement of neural plate cells, thus causing mediolateral narrowing and rostrocaudal lengthening of the neural plate (Wallingford and Harland, 2002). This cell rearrangement in the neural plate is similar to the convergent extension during gastrulation, which is regulated by Rho-GTPases under the control of the Wnt/PCP signal (Habas et al., 2001, 2003; Tada et al., 2002). Taking these findings together with our data, we think it likely that the cell rearrangement during neurulation also is regulated by the Wnt/PCP signal via Rho activation. Neural plate cells in Wnt/PCP-inhibited embryos had increased surface area (Figure 7E), perhaps due to inhibition of Rho-mediated actomyosin apical constriction. Because the Wnt/PCP signal induces both cell rearrangement and changes in the shape of individual cells in the neural plate, the two events may be linked. In fact, the orientated constriction of actomyosin that is regulated by Rho-kinase drives cell rearrangement in Drosophila germ band elongation (Bertet et al., 2004). Our data suggest that the actomyosin constriction generated by the Rho–Rho-kinase pathway also involves rearrangement of neural plate cells during neurulation.
The experimental approaches and findings we have presented will help explain the diverse physiological functions of Rho and their comprehensive features and hierarchy. In this study, we found a similar apical accumulation of Rho in multiple placodes. Because placodes, like the neural plate, are areas of thickened ectoderm that are curving and invaginating, our study is a useful starting point for further exploration of more general morphogenetic mechanisms, despite the numerous specific differences between individual cases.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Shigenobu Yonemura (yonemura{at}cdb.riken.jp)
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REFERENCES |
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