Abstract

Regulated growth and branching of dendritic processes is critical for the establishment of neuronal circuitry and normal brain functions. Rho family GTPases, including RhoA, Rac1, and Cdc42, play a prominent role in dendritic development. RhoA inhibits dendritic branching and growth, whereas Rac1/Cdc42 does the opposite. It has been suggested that the activity of RhoA must be kept low to allow dendritic growth. However, how neurons restrict the activation of RhoA for proper dendritic development is not clear. In the present study, we undertook a comprehensive loss-of-function analysis of putative RhoA GTPase-activating proteins (RhoA GAPs) in the cortical neurons. The expression of 16 RhoA GAPs was detected in the developing rat brain, and RNA interference experiments suggest that 2 of them, Myo9b and RICS, are critical regulators of dendritic morphogenesis. Knockdown of either Myo9b or RICS in cultured cortical neurons or developing cortex resulted in decreased dendrite length and number. Inhibition of RhoA/ROCK signaling restores the defects of dendritic morphology induced by knockdown of Myo9b or RICS. These data demonstrate that Myo9b and RICS repress RhoA/Rock signaling and modulate dendritic morphogenesis in cortical neurons, providing evidence for critical physiological function of RhoA GAPs in regulation of dendritic development.

Introduction

Neuronal dendrites are concerned with receiving and processing information and often highly branched. The extent of arborization of the dendritic tree correlates with the number and distribution of inputs that the neuron can receive (McAllister 2000; Jan YN and Jan LY 2001). The development of dendrites involves several discrete morphological steps, which depends on the regulation of the cytoskeleton in response to extra- or intracellular cues. Deficits in the regulation of the dendritic cytoskeleton affect both the structure and the function of dendrites and are likely to lead to cognitive impairment in some instances (Newey et al. 2005).

The small GTPases of the Rho subfamily, including RhoA, Rac1, and Cdc42, function as critical regulators of the actin cytoskeleton (Heasman and Ridley 2008; Nowak et al. 2008) to play important roles in dendritic development (Bishop and Hall 2000; Van Aelst and Cline 2004; Govek 2005). Rho GTPases cycle between an active GTP-bound state and an inactive GDP-bound state. GTP-bound Rho GTPases interact with downstream target molecules and transmit their signals. The activation state of Rho GTPases is controlled by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs stimulate the exchange of GDP for GTP to generate the activated form, while GAPs increase the rate of GTP hydrolysis to inactivate Rho GTPases (Adra et al. 1997; Van Aelst and D'Souza-Schorey 1997; Etienne-Manneville and Hall 2002; Van Aelst and Cline 2004).

Studies have demonstrated that Rac1/Cdc42 and RhoA have antagonistic effects on dendritic development. Rac1 acts as a positive regulator of dendrite growth and dynamics, whereas RhoA inhibits dendrite growth (Bishop and Hall 2000; Van Aelst and Cline 2004; Govek 2005; Koh 2006). Therefore, it has been suggested that the concurrent Rac1/Cdc42 activation and RhoA inactivation are required for normal dendritic development and functional neuronal circuitry formation (Govek 2005).- Accumulating evidences demonstrated that RacGEFs are key integrators of signaling that promote dendritic development via stimulating activation of Rac1, whereas interference the function of RacGEFs might lead to impaired dendritic development, supporting the view that Rac1 activation is required for dendritic development. However, how neurons restrict the activation of RhoA for proper dendritic development is unclear. As negative regulators of RhoA, RhoA GAPs are potential candidates that could inactivate RhoA to promote dendrite growth. It was reported that overexpression of P190 RhoGAP or P250 RhoGAP results in extensive neurite outgrowth presumably through the inactivation of RhoA (Brouns et al. 2001; Nakazawa et al. 2003). P190 inactivation leads to axon branch retraction in Drosophila mushroom body neurons (Billuart et al. 2001). The human genome is predicted to encode 59–77 GAPs for GTPases of Rho family (Lander et al. 2001; Venter et al. 2001), and an individual GAP has specificity for certain Rho GTPases. However, little is known about which GAPs inhibit RhoA in neuronal cells to regulate dendrite growth, or whether this occurs in vivo.

In the present study, we used a loss-of-function analysis to explore the role of RhoA GTPase-activating proteins (RhoA GAPs) in dendritic morphogenesis in primary cultured cortical neurons. We screened 18 potential RhoA GAPs and identified 2 of them, Myo9b and RICS, as RhoA GAPs that modulate dendrite growth. We further confirmed the function of Myo9b and RICS in the development of dendritic trees of cortical neurons in vivo. Furthermore, we show that the expression of dominant negative mutant of ROCK (ROCK DN) or treatment of Y27632, inhibitor of ROCK, could restore the defects of dendritic morphology induced by knockdown of Myo9b or RICS. Thus, our results indicate that Myo9b and RICS are RhoA GAPs critical for dendritic development of cortical neurons.

Materials and Methods

Animals

Sprague–Dawley rats were provided by SLAC laboratory Animal Co. Ltd. All animal treatments were strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Reagents and Antibodies

The following reagents and antibodies were used in this study: BLOCK-iT Pol II miR RNAi Expression Vector Kits (Invitrogen), Dulbecco’s MEM (GIBCO), Neurobasal medium (GIBCO), B27 supplement (Invitrogen), F-12 Ham (Kaighn’s modification) (Sigma), fetal bovine serum (FBS) (Hyclone), heat-inactivated horse serum (GIBCO), rabbit polyclonal antibodies against green fluorescent protein (GFP) (Invitrogen), Biotinylated anti-rabbit IgG (Jackson), Cy2-conjugated streptavidin (Jackson), TRIzol (Invitrogen), SuperScript III Reverse Transcriptase Kit (Invitrogen), SYBR premix taq (TaKaRa), trypsin (GIBCO), and Poly-D-lysine (Sigma), DIG RNA Labeling Kit (SP6/T7) (Roche), anti-DIG antibody (Fab) fragment conjugated with alkaline phosphatase (Roche).

Plasmid Constructs

The microRNA (miRNA)-based RNAi system, which uses the pcDNA 6.2-GW/EmGFP-miR expression vector to enable simultaneous expression of the EmGFP protein and miRNA, was purchased from Invitrogen. The protein level of EmGFP corresponds to the expression of miRNA. LacZ RNAi (Invitrogen) was used as a control. According to the sequences of each RhoGAP, 2 pairs of single-stranded DNA oligonucleotides were designed by Invitrogen’s RNAi Designer online (Supplementary Table 1). Plasmids encoding RhoA CA were provided by Prof. Xiaobing Yuan (Institutes of Neuroscience, Shanghai Institutes for Biological Science, Chinese Academy of Science, China). Plasmids encoding constitutively active mutant of ROCK (ROCK CA) and dominant active mutant of ROCK (ROCK DN) were provided by Prof. Kazuto Kobayashi (Fukushima Medical College, Japan) (Kobayashi et al. 2004). Plasmids encoding RICS and RICS R58I were provided by Prof. Takanobu Nakazawa and Prof. Tadashi Yamamoto (University of Tokyo, Japan) (Nakazawa et al. 2003). Plasmids encoding Myo9b and Myo9b R1695M were provided by Prof. Martin Bähler (Westfalian Wilhelms-University, Germany) (Muller et al. 1997).

RNA Isolation, Reverse Transcription, and Quantitative PCR

Total RNA was extracted from brain tissues or cells using TRIzol total RNA isolation reagent, according to the manufacturer's instructions. Complementary deoxyribonucleic acid (cDNA) was synthesized from total RNA using SuperScript III Reverse Transcriptase Kit, according to the manufacturer's instructions. The reactions were incubated in a water bath for 15 min at 37 °C, 5 s at 85 °C, and then held at 4 °C. Quantitative PCR was performed using an SYBR premix taq system (TaKaRa) with specific primers designed for each RhoA GAP. The relative expression level was calculated and normalized to the endogenous reference control gene GAPDH.

Cell Culture and Transfection

C6 cells were maintained in F-12 Ham (Kaighn’s modification) supplement with 15% horse serum and 2.5% FBS. Cells were electroporated with miRNA-based RNAi plasmids by nucleofection as described by the manufacturer (Amaxa). Briefly, 2 × 106 cells were suspended with 2 μg DNA in the nucleofection solution provided and were electroporated by nucleofection. Cultures of cortical neurons were prepared from Sprague–Dawley rats of postnatal day 0 (P0). After treatment with 0.1 mg/mL trypsin (GIBCO) and 0.6 μg/mL DNase (Sigma) for 15 min at 37 °C and mechanical dissociation, cortical cells were electroporated by nucleofection as described by the manufacturer (Amaxa). Briefly, 4 × 106 neurons were suspended, with 4 μg DNA in the nucleofection solution provided. Cells were plated at a final density of 2 × 105/cm2 on coverslips coated with Poly-D-lysine and allowed to recover in Dulbecco's Modified Eagle's Medium (Invitrogen) with 10% calf serum for 2 h before replacement with Neurobasal medium containing 2 mM glutaMAX, 1% penicillin–streptomycin, and B27 supplement (Invitrogen). For image quantitation, neurons were fixed 96 h later. Morphology of neurons was visualized by GFP fluorescence.

In Utero Electroporation

Plasmids were transfected using intraventricular injection followed by in utero electroporation (Kawauchi et al. 2003, 2006; Wang et al. 2007). In brief, Sprague–Dawley rats at 15.5 days of gestation were anesthetized with 10% chloral hydrate (4 mL/kg body weight). One to two microliter (4 μg/μL) miRNA-based RNAi plasmids mixed with Fast Green (2 mg/mL; Sigma) were injected by transuterus pressure microinjection into the lateral ventricle of embryos. Then, electric pulses generated by the ElectroSquireportator T830 (BTX) were applied to the cerebral wall at 5 repeats of 60 V for 50 ms with an interval of 950 ms.

Immunocytochemistry

For immunostaining, brains of rats that had been electroporated were removed at postnatal day 3 (P3) and fixed in 4% paraformaldehyde over night after transcardial perfusion. After dehydration in 20% glucose, brains were cut at coronal cryostat sections of 50 μm. Sections were processed for immunostaining by a 3-step free-floating protocol. Primary antibody was diluted in blocking solution containing 0.3% Triton X-100 and 5% goat serum. For GFP staining, brain sections were incubated with rabbit anti-GFP for 2 h at room temperature. Biotinylated anti-rabbit IgG and then Cy2-conjugated streptavidin were performed at room temperature for 1 h.

Image Acquisition and Quantitation

Images were obtained using Carl Zeiss LSM 510 confocal microscope with 40× [numerical aperture = 1.3] objective. All morphology measurements were made on individual Z-stack images of segments. GFP-expressing neurons were randomly selected and imaged. For quantification, a dendrite or a dendritic process of neurons at days in vitro (DIV) 2 is defined as a protrusion whose length is longer than half of the diameter of a cell body. The longest protrusion from a cell body, recognized as an axonal process, was excluded from dendritic process and dendrite quantification. Image analysis of brain sections was performed as described previously (Chen et al. 2008, 2011). For quantitative analysis, electroporation was performed targeting the same region of the developing cortex. Neuronal morphology was traced and analyzed by Neurolucida (MBF Bioscience).

In Situ Hybridization

A partial rat cDNA fragment (corresponding to nucleotides 5795–6234 in rat RICS cDNA or nucleotides 3530–3982 in rat Myo9b cDNA) was obtained by reverse-transcription polymerase chain reaction and cloned into pcDNA3.0. After verification of the sequence, Digoxygenin (dig)-labeled RNA probes were generated using DIG RNA Labeling Kit (SP6/T7), according to the manufacturer's instructions.

The free-floating slice hybridization method (Landry et al. 1997) was employed. Briefly, the frozen coronal slices of 30 μm thickness were sectioned on freezing microtome. Slices were permeated in 0.1% Tween-20 in phosphate-buffered saline (PBS), fixed in 4% PBS-buffered paraformaldehyde solution, and digested with 2 μg/mL proteinase K for 10 min at room temperature. After the cessation of digestion, hybridization was performed at 37 °C (RICS) or 45 °C (Myo9b) for 18 h in hybridization solution containing 50% formamide (v/v) deionized, 5×SSC, 0.02% SDS (w/v), 2% blocking solution (Roche) (v/v), and 100–150 ng/250 μL dig-labeled probes. Following hybridization, slices were washed 3 times in 1×SSC for 30 min and once in 0.1×SSC for 15 min at 60 °C (RICS) or 70 °C (Myo9b). Immunological detection of digoxygenin was carried out by incubation of the slices with anti-DIG antibody (Fab) fragment conjugated with alkaline phosphatase (1:200) and 5% sheep serum at room temperature for 4 h. And then the slices were developed with NTB/BCIP Stock Solution (Roche) at room temperature for 2–4 h.

RNA Interference and Rescue

Cortical neurons were isolated and electroporated with miRNA-based RNAi plasmids by nucleofection as described by the manufacturer (Amaxa). The cDNA encoding RICS or ROCK DN was cotransfected with miRNA-based RNAi plasmids. The neuronal morphology was analyzed 96 h after transfection.

Statistical Analysis

Values were presented as mean ± standard error. In most cases, multiple comparison of groups was performed by one-way analysis of variance (ANOVA) followed by a Fisher’s least significant difference (LSD) post hoc test. Student’s t-test was used to measure significance of differences between 2 groups. Differences with P < 0.05 were considered as significant.

Results

RhoA/ROCK Negatively Regulates Dendritic Development of Cortical Neurons

To characterize the function of RhoA/ROCK signaling in dendritic development, constitutively active mutant of RhoA (RhoA CA) and constitutively active mutant of ROCK (ROCK CA) were transfected into rat cortical neurons. Overexpression of RhoA CA dramatically inhibited dendrite initiation from cell bodies, and many cell bodies remained round shaped with fewer membrane protrusions after 2 DIV (Supplementary Fig. 1A). Overexpression of ROCK CA mimicked the phenotype in RhoA CA overexpressing neurons (Supplementary Fig. 1A). Quantification analysis reveals that the total dendritic process number and length were significantly decreased in neurons expressing RhoA CA or ROCK CA compared with the neurons expressing control protein (P < 0.001) (Supplementary Fig. 1B). In contrast, cortical neurons treated with ROCK inhibitor Y27632 (30 μM) exhibited more and longer dendritic processes at DIV 2 (Supplementary Fig. 1A), and the total length and number of dendritic process were markedly increased (Supplementary Fig. 1B). These results are consistent with previous findings that RhoA/ROCK signaling negatively regulates dendritic development (Nakayama et al. 2000).

We next examined the effect of increase of RhoA activity on dendritic morphogenesis in developing cortical neurons in vivo using in utero electroporation approach (Kawauchi et al. 2003, 2006; Wang et al. 2007). Expression of RhoA CA inhibited dendritic development of cortical neurons located at layer II/III (Supplementary Fig. 1C). Analysis of neuronal morphology indicated a 44% reduction of dendrite number and 28% reduction of dendrite length in neurons expressing RhoA CA compared with control neurons (Supplementary Fig. 1D). These results suggest that restricted RhoA activation is required for normal dendritic development in vivo.

Expression of Putative RhoA GAPs in Cerebral Cortex during Development

To understand how neurons confine the activation of RhoA for proper dendritic development, we undertook a comprehensive loss-of-function analysis of the predicted RhoA GAPs. Eighteen potential RhoA GAPs that have been reported to be able to target RhoA in vitro or in vivo (Moon and Zheng 2003; Bos et al. 2007; Tcherkezian and Lamarche-Vane 2007) were examined. The expression of these potential RhoA GAPs in rat cerebral cortex at E16, E18, P0, P3, P7, P14, and P60 were analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Of the 18 RhoA GAPs, relative mRNA levels of RICS, GRAF, GRAF2, DLC1, DLC3, and ARAP2 increased during development (Table 1 and Fig. 1A), whereas Myo9a, Myo9b, GMIP, P50, and P190A decreased (Table 1 and Fig. 1B). Among the decreased GAPs, GMIP, P50, and P190A markedly rose around E18 and then declined (Table 1 and Fig. 1B). The transcript level of ARAP1 and DLC2 did not change during the period of rat development (Table 1 and Fig. 1C). The level of Oligophrenin1 and PARG1 mRNA reached peak around P0 and then recovered to the level as E16 (Table 1 and Fig. 1D). There was a trend of decrease for the level of P190B at P0, and then it gradually increased (Table 1 and Fig. 1D). SrGAP1 and P115 transcripts were not detected in rat cerebral cortex (Table 1).

Table 1

Relative mRNA levels of 18 putative RhoA GAPs in developing rat cerebral cortex

Gene Gene ID Relative mRNA levela (mean ± SE) 
E16 E18 P0 P3 P7 P14 
DLC1 58834 0.559 ± 0.049 0.694 ± 0.078 0.723 ± 0.075 1.151 ± 0.138** 1.090 ± 0.155** 1.277 ± 0.111*** 
DLC2 498130 0.157 ± 0.020 0.171 ± 0.030 0.094 ± 0.010* 1.128 ± 0.029 0.082 ± 0.011* 0.111 ± 0.012 
DLC3 312113 0.173 ± 0.019 0.190 ± 0.025 0.161 ± 0.011 0.333 ± 0.069 0.280 ± 0.038 0.894 ± 0.109*** 
Myo9a 171296 5.470 ± 1.150 7.736 ± 2.307 4.205 ± 0.349 4.975 ± 0.684 4.181 ± 0.360 1.887 ± 0.465* 
Myo9b 25486 4.040 ± 0.612 4.659 ± 0.983* 2.648 ± 0.060* 3.248 ± 0.184 1.476 ± 0.137*** 1.478 ± 0.319*** 
GRAF 307459 0.539 ± 0.070 1.202 ± 0.242 2.124 ± 0.088* 2.720 ± 0.231*** 2.912 ± 0.203*** 5.186 ± 0.968*** 
GRAF2 688429 0.328 ± 0.040 0.475 ± 0.039 0.474 ± 0.038 0.521 ± 0.056* 0.454 ± 0.060 0.846 ± 0.066*** 
RICS 315530 1.212 ± 0.101 1.907 ± 0.251* 2.229 ± 0.143 2.573 ± 0.138 2.180 ± 0.216 2.983 ± 0.178*** 
ARAP1 361617 0.394 ± 0.012 0.450 ± 0.038 0.474 ± 0.055 0.621 ± 0.054** 0.417 ± 0.037 0.470 ± 0.081 
ARAP2 305367 0.166 ± 0.038 0.575 ± 0.155 1.195 ± 0.134* 2.590 ± 0.237*** 2.473 ± 0.292*** 2.915 ± 0.430*** 
Oligophrenin1 312108 1.493 ± 0.146 2.184 ± 0.439* 2.420 ± 0.111** 1.974 ± 0.151 1.193 ± 0.198 1.250 ± 0.185 
P190A 306400 11.464 ± 1.535 15.449 ± 0.909* 15.217 ± 1.285* 13.992 ± 0.584 11.599 ± 1.010 5.984 ± 0.752*** 
P190B 299012 3.643 ± 0.656 3.969 ± 1.201 2.250 ± 0.365 3.970 ± 0.489 4.404 ± 0.286 4.719 ± 0.454 
P115 246249 ND ND ND ND ND ND 
GMIP 306357 1.950 ± 0.336 3.418 ± 0.556*** 2.421 ± 0.159 1.850 ± 0.189 0.912 ± 0.073* 0.256 ± 0.052*** 
P50 311193 4.529 ± 0.398 7.188 ± 0.650** 6.790 ± 0.544* 5.978 ± 1.068 4.610 ± 0.285 2.588 ± 0.227* 
PARG1 310833 0.587 ± 0.056 1.986 ± 0.494*** 1.841 ± 0.213** 1.801 ± 0.282** 1.291 ± 0.119* 0.705 ± 0.083 
SrGAP1 314903 ND ND ND ND ND ND 
Gene Gene ID Relative mRNA levela (mean ± SE) 
E16 E18 P0 P3 P7 P14 
DLC1 58834 0.559 ± 0.049 0.694 ± 0.078 0.723 ± 0.075 1.151 ± 0.138** 1.090 ± 0.155** 1.277 ± 0.111*** 
DLC2 498130 0.157 ± 0.020 0.171 ± 0.030 0.094 ± 0.010* 1.128 ± 0.029 0.082 ± 0.011* 0.111 ± 0.012 
DLC3 312113 0.173 ± 0.019 0.190 ± 0.025 0.161 ± 0.011 0.333 ± 0.069 0.280 ± 0.038 0.894 ± 0.109*** 
Myo9a 171296 5.470 ± 1.150 7.736 ± 2.307 4.205 ± 0.349 4.975 ± 0.684 4.181 ± 0.360 1.887 ± 0.465* 
Myo9b 25486 4.040 ± 0.612 4.659 ± 0.983* 2.648 ± 0.060* 3.248 ± 0.184 1.476 ± 0.137*** 1.478 ± 0.319*** 
GRAF 307459 0.539 ± 0.070 1.202 ± 0.242 2.124 ± 0.088* 2.720 ± 0.231*** 2.912 ± 0.203*** 5.186 ± 0.968*** 
GRAF2 688429 0.328 ± 0.040 0.475 ± 0.039 0.474 ± 0.038 0.521 ± 0.056* 0.454 ± 0.060 0.846 ± 0.066*** 
RICS 315530 1.212 ± 0.101 1.907 ± 0.251* 2.229 ± 0.143 2.573 ± 0.138 2.180 ± 0.216 2.983 ± 0.178*** 
ARAP1 361617 0.394 ± 0.012 0.450 ± 0.038 0.474 ± 0.055 0.621 ± 0.054** 0.417 ± 0.037 0.470 ± 0.081 
ARAP2 305367 0.166 ± 0.038 0.575 ± 0.155 1.195 ± 0.134* 2.590 ± 0.237*** 2.473 ± 0.292*** 2.915 ± 0.430*** 
Oligophrenin1 312108 1.493 ± 0.146 2.184 ± 0.439* 2.420 ± 0.111** 1.974 ± 0.151 1.193 ± 0.198 1.250 ± 0.185 
P190A 306400 11.464 ± 1.535 15.449 ± 0.909* 15.217 ± 1.285* 13.992 ± 0.584 11.599 ± 1.010 5.984 ± 0.752*** 
P190B 299012 3.643 ± 0.656 3.969 ± 1.201 2.250 ± 0.365 3.970 ± 0.489 4.404 ± 0.286 4.719 ± 0.454 
P115 246249 ND ND ND ND ND ND 
GMIP 306357 1.950 ± 0.336 3.418 ± 0.556*** 2.421 ± 0.159 1.850 ± 0.189 0.912 ± 0.073* 0.256 ± 0.052*** 
P50 311193 4.529 ± 0.398 7.188 ± 0.650** 6.790 ± 0.544* 5.978 ± 1.068 4.610 ± 0.285 2.588 ± 0.227* 
PARG1 310833 0.587 ± 0.056 1.986 ± 0.494*** 1.841 ± 0.213** 1.801 ± 0.282** 1.291 ± 0.119* 0.705 ± 0.083 
SrGAP1 314903 ND ND ND ND ND ND 

Note: ND, not detected; SE, standard error.

a

Relative mRNA levels of 18 RhoA GAPs in rat cerebral cortex were quantified by qRT-PCR and normalized as a percentage of GADPH.

*P < 0.05, **P < 0.01, ***P < 0.001 versus data from E16, n = 4 for each time point, one-way ANOVA followed by a Fisher’s LSD post hoc test.

Figure 1.

Relative mRNA levels of 18 putative RhoA GAPs in the developing rat cerebral cortex. Rats at E16, E18, P0, P3, P7, P14, and P60 were sacrificed, and the cerebral cortex of their brains were obtained for qRT-PCR. The x-axis indicates the days after birth, defining P0 as 0, days before birth as negative values, and days after birth as positive values. The y-axis shows the relative mRNA levels as a percentage of GADPH. Relative mRNA levels at P14 were compared with those at E16, and those increased (A), decreased (B), unchanged (C), or with a peak or valley expression (D) was plotted.

Figure 1.

Relative mRNA levels of 18 putative RhoA GAPs in the developing rat cerebral cortex. Rats at E16, E18, P0, P3, P7, P14, and P60 were sacrificed, and the cerebral cortex of their brains were obtained for qRT-PCR. The x-axis indicates the days after birth, defining P0 as 0, days before birth as negative values, and days after birth as positive values. The y-axis shows the relative mRNA levels as a percentage of GADPH. Relative mRNA levels at P14 were compared with those at E16, and those increased (A), decreased (B), unchanged (C), or with a peak or valley expression (D) was plotted.

Knockdown of Myo9a, Myo9b, or RICS Inhibits Dendritic Morphogenesis in Cultured Cortical Neurons

We generated 2 miRNA plasmids targeting distinct sequences for each RhoA GAPs. The knockdown efficiency of miRNA was examined in Rat C6 cell line (Supplementary Table 1). Rat cortical neurons were transfected with plasmids encoding control or RhoA GAP miRNA (target #1), and neuronal morphology was analyzed at DIV 4. Transfected neurons were stained with antibody recognizing GFP as morphological marker.

As shown in Fig. 2A,B, of the 16 different RhoA GAPs, knockdown of Myo9a, Myo9b, or RICS reduced both total dendrite number and length. Morphology analysis showed that expression of Myo9b miRNA in cultured neurons resulted in a marked reduction in the total dendrite length (58.2 ± 8.6% of control, P < 0.001) and number (57.4 ± 7.8% of control, P < 0.001) as compared with control miRNA expressing neurons (Fig. 2A). Similarly, transfection of RICS miRNA decreased total dendrite length and number by approximately 40% (Fig. 2A). Neurons expressing Myo9a miRNA also showed reduced total dendrite length and number, but at a relative moderate level as compared with neurons expressing Myo9b or RICS miRNA (Fig. 2A). Neurons expressing ARAP2 miRNA showed milder reduction in total dendrite length but not in total dendrite number, and neurons expressing P190A miRNA exhibited obvious reduction in total dendrite number but not in total dendrite length. We thus focused on Myo9a, Myo9b, or RICS for further examination. To excluding out the possible off-target effects of individual miRNA, we used the second set of miRNA plasmids (miRNA #2) targeting distinct sequence of Myo9a, Myo9b, or RICS. Morphology analysis revealed that the 2 sets of miRNAs for Myo9a, Myo9b, and RICS exhibited similar effect on dendritic morphogenesis (Fig. 2C). Furthermore, the defects of dendrite morphology induced by knockdown of RICS in rat cortical neurons could be rescued by cotransfection of resistant human RICS (Supplementary Fig. S3).

Figure 2.

Identification of Myo9a, Myo9b, and RICS as essential RhoGAPs in the regulation of dendritic morphogenesis of cultured neurons. (A, B) Cortical neurons transfected with control miRNA plasmid or miRNA plasmid of target #1 of the indicated RhoA GAPs were fixed and stained with antibody against GFP at DIV 4. (A) Total dendrite number and length of GFP-positive neurons were quantified. Data are presented as mean ± standard error (SE) (n ≥ 30 neurons for each group). *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by a Fisher’s LSD post hoc test. (B) Representative images are shown. Scale bar, 10 μm. (C) Quantification of total dendrite number and length of cortical neurons transfected with control miRNA plasmid or miRNA plasmid of target #1 or #2 of indicated RhoA GAPs. Data are presented as mean ± SE (n ≥ 40 neurons for each group). *P < 0.05, **P < 0.01, *** P < 0.001, one-way ANOVA followed by a Fisher’s LSD post hoc test.

Figure 2.

Identification of Myo9a, Myo9b, and RICS as essential RhoGAPs in the regulation of dendritic morphogenesis of cultured neurons. (A, B) Cortical neurons transfected with control miRNA plasmid or miRNA plasmid of target #1 of the indicated RhoA GAPs were fixed and stained with antibody against GFP at DIV 4. (A) Total dendrite number and length of GFP-positive neurons were quantified. Data are presented as mean ± standard error (SE) (n ≥ 30 neurons for each group). *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by a Fisher’s LSD post hoc test. (B) Representative images are shown. Scale bar, 10 μm. (C) Quantification of total dendrite number and length of cortical neurons transfected with control miRNA plasmid or miRNA plasmid of target #1 or #2 of indicated RhoA GAPs. Data are presented as mean ± SE (n ≥ 40 neurons for each group). *P < 0.05, **P < 0.01, *** P < 0.001, one-way ANOVA followed by a Fisher’s LSD post hoc test.

Knockdown of Myo9b or RICS Inhibits Dendritic Morphogenesis In Vivo

We next explored the importance of Myo9a, Myo9b, and RICS in dendritic morphogenesis in vivo using in utero electroporation. Myo9a, Myo9b, and RICS miRNA plasmids were electroporated into the lateral ventricles of embryonic rats at E15.5, and the morphogenesis of transfected cortical neurons was evaluated at P3. Cortical neurons expressing Myo9a, Myo9b, or RICS miRNA were able to migrate into cortical plate as control miRNA expressing neurons did. Morphology analysis of cortical neurons at layer II/III showed that knockdown of Myo9b (Fig. 3A,B) or RICS (Fig. 3C,D) significantly simplified the dendritic arborization, as indicated by the reduced dendrite length and number. Similar results were obtained using the second set of Myo9b or RICS miRNAs (Fig. 3). However, significant morphological change was not observed in neurons expressing Myo9a miRNA in the cortex as compared with neurons expressing control miRNA (Supplementary Fig. 2A,B). These results demonstrate that Myo9b and RICS are necessary for normal dendritic morphogenesis of cortical neurons in vivo.

Figure 3.

Knockdown of Myo9b or RICS inhibits dendritic morphogenesis in the developing cerebral cortex. (A, C) Representative images of cortical neurons in layer II/III transfected with control miRNA or miRNA of target #1 or #2 of Myo9b (A) or RICS (C). Indicated plasmids were electroporated into E15.5 rat embryonic brains in utero, and cortical neurons were stained at P3 with GFP antibody. Scale bar, 10 μm. (B, D) Quantification of total dendrite number and length of GFP-positive neurons as indicated in (A, C). Data are presented as mean ± standard error (n ≥ 40 neurons from 4 to 5 brains). **P < 0.01, ***P < 0.001, one-way ANOVA followed by a Fisher’s LSD post hoc test.

Figure 3.

Knockdown of Myo9b or RICS inhibits dendritic morphogenesis in the developing cerebral cortex. (A, C) Representative images of cortical neurons in layer II/III transfected with control miRNA or miRNA of target #1 or #2 of Myo9b (A) or RICS (C). Indicated plasmids were electroporated into E15.5 rat embryonic brains in utero, and cortical neurons were stained at P3 with GFP antibody. Scale bar, 10 μm. (B, D) Quantification of total dendrite number and length of GFP-positive neurons as indicated in (A, C). Data are presented as mean ± standard error (n ≥ 40 neurons from 4 to 5 brains). **P < 0.01, ***P < 0.001, one-way ANOVA followed by a Fisher’s LSD post hoc test.

Myo9b and RICS Modulate Dendritic Morphogenesis via Repressing RhoA/ROCK Signaling

We next explored mechanisms of Myo9b and RICS-regulated dendritic morphogenesis. As shown in Fig. 4A, the expression of Myo9b and RICS was detected in the cortical plate (CP) region of developing brain at P3 using in situ hybridization with specific antisense cRNA of Myo9b or RICS. The effects of overexpressing Myo9b and RICS on dendritic morphogenesis were further examined in cortical neurons at DIV 2. Overexpression of Myo9b or RICS significantly increased the length and number of dendritic processes, whereas overexpression of GAP activity-deficient mutants of Myo9b or RICS (Myo9b R1695M or RICS R58I) had no such effects (Fig. 4B,C). These results suggest the involvement of the GAP activity of Myo9b and RICS in regulation of dendritic morphogenesis. To examine whether the reduced dendritic complexity induced by knockdown of Myo9b or RICS is a result of overactivation of RhoA/ROCK signaling, cortical neurons transfected with Myo9b or RICS miRNA were treated with Y27632 or cotransfected with ROCK DN and analyzed at DIV 4. Results of quantification of total dendrite number and length showed that the inhibitory effects of Myo9b and RICS miRNA on dendritic morphogenesis could be rescued by either Y27632 treatment or ROCK DN overexpression (Fig. 4D,E). These results suggest that Myo9b and RICS may regulate dendritic morphogenesis through repressing RhoA/ROCK signaling. Furthermore, compared with cortical neurons transfected with either Myo9b miRNA or RICS miRNA, cortical neurons cotransfected with both Myo9b and RICS miRNAs exhibited shorter dendrite length and less dendrite number, suggesting additive effects (Fig. 4F,G). Taken together, these results indicate that Myo9b and RICS regulate dendritic morphogenesis, which may function through repressing RhoA/ROCK signaling.

Figure 4.

Myo9b and RICS modulate dendritic morphogenesis via repressing RhoA/ROCK signaling. (A) In situ hybridization for Myo9b and RICS in slices of P3 brain. High magnification views of the cortex enclosed by rectangles are shown right. CP, cortical plate; IZ, intermedial zone; SVZ/VZ, subventricular zone/ventricular zone. Scale bar, 50 μm. (B) Representative images of cortical neurons transfected with indicated plasmids. Neurons at DIV 2 were stained with antibody against GFP. (C) Quantification of total dendritic process number and length of GFP-positive neurons as indicated in (B). Data are presented as mean ± standard error (SE) (n ≥ 37 neurons for each group). ***P < 0.001, one-way ANOVA followed by a Fisher’s LSD post hoc test. (D) Representative images of cortical neurons transfected with indicated plasmids or treated with Y27632. DIV 4 neurons were stained with antibody against GFP. (E) Quantification of total dendrite number and length of GFP-positive neurons as indicated in (D). Data are presented as mean ± SE (n ≥ 40 neurons for each group). #P values are relative to Ctrl i. #P < 0.05, ##P < 0.01, ###P < 0.001. *P values are relative to RICS i or Myo9b i, respectively. ***P < 0.001. One-way ANOVA followed by a Fisher’s LSD post hoc test. (F) Representative images of cortical neurons transfected with plasmids encoding Myo9b miRNA, RICS miRNA, or both. (G) Quantification of total dendrite number and length of GFP-positive neurons as indicated in (F). Data are presented as mean ± SE (n ≥ 30 neurons for each group). *P values are relative to Ctrl i. ***P < 0.001. #P values are relative to Myo9b i plus RICS i. #P < 0.05, ##P < 0.01. One-way ANOVA followed by a Fisher’s LSD post hoc test.

Figure 4.

Myo9b and RICS modulate dendritic morphogenesis via repressing RhoA/ROCK signaling. (A) In situ hybridization for Myo9b and RICS in slices of P3 brain. High magnification views of the cortex enclosed by rectangles are shown right. CP, cortical plate; IZ, intermedial zone; SVZ/VZ, subventricular zone/ventricular zone. Scale bar, 50 μm. (B) Representative images of cortical neurons transfected with indicated plasmids. Neurons at DIV 2 were stained with antibody against GFP. (C) Quantification of total dendritic process number and length of GFP-positive neurons as indicated in (B). Data are presented as mean ± standard error (SE) (n ≥ 37 neurons for each group). ***P < 0.001, one-way ANOVA followed by a Fisher’s LSD post hoc test. (D) Representative images of cortical neurons transfected with indicated plasmids or treated with Y27632. DIV 4 neurons were stained with antibody against GFP. (E) Quantification of total dendrite number and length of GFP-positive neurons as indicated in (D). Data are presented as mean ± SE (n ≥ 40 neurons for each group). #P values are relative to Ctrl i. #P < 0.05, ##P < 0.01, ###P < 0.001. *P values are relative to RICS i or Myo9b i, respectively. ***P < 0.001. One-way ANOVA followed by a Fisher’s LSD post hoc test. (F) Representative images of cortical neurons transfected with plasmids encoding Myo9b miRNA, RICS miRNA, or both. (G) Quantification of total dendrite number and length of GFP-positive neurons as indicated in (F). Data are presented as mean ± SE (n ≥ 30 neurons for each group). *P values are relative to Ctrl i. ***P < 0.001. #P values are relative to Myo9b i plus RICS i. #P < 0.05, ##P < 0.01. One-way ANOVA followed by a Fisher’s LSD post hoc test.

Discussion

Of the Rho GTPase family members, RhoA, Rac1, and cdc42 have been characterized as key molecules that link surface receptors to the organization of the actin cytoskeleton to regulate dendritic morphogenesis (Govek 2005; Newey et al. 2005). The interplay between the Rho GTPases determines the complexity of the neuronal dendrite arbor tree. Inactivation of RhoA is particularly crucial for dendritic outgrowth and arborization, demonstrated by manipulating of RhoA activity experiments in both invertebrate and in vertebrate model systems (Lee et al. 2000; Wong et al. 2000; Ahnert-Hilger et al. 2004). In our system, constitutively active RhoA dramatically simplifies dendrite tree of cortical neurons both in vitro and in vivo. Furthermore, activation or inhibition of the downstream effector ROCK leads to the similar or opposite phenotype. These results demonstrate the key role of RhoA/ROCK signaling in normal dendrite development and also indicate the importance of suppression of RhoA activity in this process. Maintenance of low RhoA activity is required for dendritic growth. However, the negative regulators of RhoA activity in vivo have not been identified. In this work, we found that Myo9b and RICS are RhoA GAPs essential for regulation of RhoA/ROCK signaling to control dendritic morphogenesis. Our data show that knockdown of Myo9b or RICS significantly decreased dendritic elongation and branching of cortical neurons and thus can be restored by overexpression of ROCK DN or inhibition of ROCK activity. These results uncover novel roles for Myo9b and RICS in neuronal dendrite development. In light of the current study, it is likely that the RhoA/ROCK pathway in cortical neurons is actively repressed by Myo9b and RICS to allow dendritic growth to occur under physiological conditions.

In neuronal systems, RhoGAPs are involved in regulating multiple processes in the morphological development of neurons (Takei et al. 2000; Billuart et al. 2001; Reczek and Bretscher 2001; Wong et al. 2001). Oligophrenin1, a RhoGAP family member that acts on multiple Rho GTPases was found to be associated with X-linked mental retardation (Billuart et al. 1998). Studies of P190 RhoGAP show that its oversynthesis induces neurite formation in a neuronal cell line (Brouns et al. 2001). Interestingly, in our study, a reduction of dendritic number induced by downregulation of P190A in cortical neurons was also observed, implying the functional role of P190A in neurite initiation and dendrite branching. However, we did not detect obvious reduction of total dendritic length after downregulation of P190A, suggesting that P190A may specifically regulate dendritic branching.

Both Myo9a and Myo9b belong to human class IX of myosin molecules, which comprise a GAP domain in the tail region in addition to their myosin head domain (Post et al. 1998). There are relatively few reports about the physiological functions of this class Myosin. Mice lacking Myo9a have been reported to develop severe hydrocephalus (Abouhamed et al. 2009). The expression of Myo9b in developing brain has been reported (Chieregatti et al. 1998), but its physiological function in the nervous system is not understood. Our study shows that Myo9b is able to regulate the dendritic morphogenesis of cortical neurons via repressing RhoA/ROCK signaling in cultures and in developing rat brain. These results suggest that Myo9b is a critical negative regulator of RhoA/ROCK signaling in cortical neurons. Recent study in macrophages reveals that Myo9b could control cell shape and motility by inhibiting RhoA, suggesting Myo9b as the most critical regulator of RhoA in macrophage (Hanley et al. 2010). Thus, Myo9b may play important roles in vivo via repressing RhoA activity. In contrast to Myo9b, we did not detect any significant effect on dendritic development in cortical neurons after knockdown of Myo9a in the developing rat brain, although Myo9a did regulate dendritic morphogenesis in cultured cortical neurons. The different phenotype after knockdown of Myo9a in vitro and in vivo may result from functional compensation of Myo9a by Myo9b in the developing rat brain. Another possible explanation could be that external cues of neurons in vivo, such as enriched neurotropic factors, are needed to activate specific RhoA GAPs to promote dendrite growth.

RICS, also identified as Grit/P200RhoGAP/P250GAP/GCGAP (Nakamura et al. 2002; Moon et al. 2003; Okabe et al. 2003; Zhao et al. 2003), is enriched in the brain and is able to promote the hydrolysis of GTP bound to RhoA, Cdc42, and Rac1 in vitro (Moon et al. 2003; Nakazawa et al. 2003; Okabe et al. 2003). As a RhoGAP, RICS exhibits functional roles in spine morphogenesis, neurite outgrowth, and axon guidance (Nakamura et al. 2002; Nakazawa et al. 2003, 2008; Okabe et al. 2003; Nasu-Nishimura et al. 2006; Chagnon et al. 2010). Interestingly, the effects of RICS on neuronal morphology seem completely dependent on its GAP function. For example, expression of RICS in Neuro-2A cells suppresses neurite outgrowth after serum withdrawal, while in the presence of serum the effect is opposite, which may result from the suppression of Cdc42 or RhoA activity in different culture condition (Nakazawa et al. 2003). Our data show that knockdown of RICS reduces dendrite complexity of cortical neurons, which is rescued by overexpression of ROCK DN and treatment of Y27632, suggesting that RICS suppresses RhoA signaling in regulation of dendrite development in cortical neurons. However, it has been reported that RICS−/− neurons at DIV 1.5 bore longer neuritis rather than shorter neuritis, and the activity of Cdc42 is higher in RICS−/− neurons than those from wild-type mice (Nasu-Nishimura et al. 2006). It is likely that RICS functions as distinct GAP for distinct Rho GTPases in the different development stages. It has been reported that in DIV 13 neurons, RICS is involved in N-methyl-D-aspartate receptor-mediated RhoA activity (Nakazawa et al. 2008), while in neurons at DIV 7, it acts on Rac1 and induces a different phenotype (Impey et al. 2010).

Human genome analysis predicts about 80 RhoGAPs in Homo sapiens, which is far outnumbering the Rho GTPases they regulate (Moon and Zheng 2003). Each RhoGAPs might play a specialized role in regulating the activity of individual Rho GTPase and their specific functions, since many RhoGAPs have specific distribution and expression pattern in multicellular organisms. Our systematical analysis provides a novel insight into the physiological functions of RhoA GAPs in the dendritic development and further support the view that restricted RhoA/ROCK signaling is required for normal dendritic development.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/

Funding

Ministry of Science and Technology (grant 2009CB522006 to L.M.) and National Natural Science Foundation of China (grants 30830042 and 31121061 to L.M., 30800646 to H.L., and 30900852 to Y.C.).

We thank Dr X. Yuan for providing RhoA CA, Dr K. Kobayashi for ROCK DN, Dr M. Bählerb for Myo9b and Myo9b R1695M, Drs T. Nakazawa and T. Yamamoto for RICS and RICS R58I, Drs F. Wang and Z. Wu for comments and technical support. Conflict of Interest : None declared.

References

Abouhamed
M
Grobe
K
San
IV
Thelen
S
Honnert
U
Balda
MS
Matter
K
Bahler
M
Myosin IXa regulates epithelial differentiation and its deficiency results in hydrocephalus
Mol Biol Cell
 , 
2009
, vol. 
20
 (pg. 
5074
-
5085
)
Adra
CN
Manor
D
Ko
JL
Zhu
S
Horiuchi
T
Van Aelst
L
Cerione
RA
Lim
B
RhoGDIgamma: a GDP-dissociation inhibitor for Rho proteins with preferential expression in brain and pancreas
Proc Natl Acad Sci U S A
 , 
1997
, vol. 
94
 (pg. 
4279
-
4284
)
Ahnert-Hilger
G
Holtje
M
Grosse
G
Pickert
G
Mucke
C
Nixdorf-Bergweiler
B
Boquet
P
Hofmann
F
Just
I
Differential effects of Rho GTPases on axonal and dendritic development in hippocampal neurones
J Neurochem
 , 
2004
, vol. 
90
 (pg. 
9
-
18
)
Billuart
P
Bienvenu
T
Ronce
N
des Portes
V
Vinet
MC
Zemni
R
Roest Crollius
H
Carrie
A
Fauchereau
F
Cherry
M
, et al.  . 
Oligophrenin-1 encodes a rhoGAP protein involved in X-linked mental retardation
Nature
 , 
1998
, vol. 
392
 (pg. 
923
-
926
)
Billuart
P
Winter
CG
Maresh
A
Zhao
X
Luo
L
Regulating axon branch stability: the role of p190 RhoGAP in repressing a retraction signaling pathway
Cell
 , 
2001
, vol. 
107
 (pg. 
195
-
207
)
Bishop
AL
Hall
A
Rho GTPases and their effector proteins
Biochem J
 , 
2000
, vol. 
348
 
Pt 2
(pg. 
241
-
255
)
Bos
JL
Rehmann
H
Wittinghofer
A
GEFs and GAPs: critical elements in the control of small G proteins
Cell
 , 
2007
, vol. 
129
 (pg. 
865
-
877
)
Brouns
MR
Matheson
SF
Settleman
J
p190 RhoGAP is the principal Src substrate in brain and regulates axon outgrowth, guidance and fasciculation
Nat Cell Biol
 , 
2001
, vol. 
3
 (pg. 
361
-
367
)
Chagnon
MJ
Wu
CL
Nakazawa
T
Yamamoto
T
Noda
M
Blanchetot
C
Tremblay
ML
Receptor tyrosine phosphatase sigma (RPTPsigma) regulates, p250GAP, a novel substrate that attenuates Rac signaling
Cell Signal
 , 
2010
, vol. 
22
 (pg. 
1626
-
1633
)
Chen
G
Sima
J
Jin
M
Wang
KY
Xue
XJ
Zheng
W
Ding
YQ
Yuan
XB
Semaphorin-3A guides radial migration of cortical neurons during development
Nat Neurosci
 , 
2008
, vol. 
11
 (pg. 
36
-
44
)
Chen
Y
Wang
F
Long
H
Wu
Z
Ma
L
GRK5 promotes F-actin bundling and targets bundles to membrane structures to control neuronal morphogenesis
J Cell Biol
 , 
2011
, vol. 
194
 (pg. 
905
-
920
)
Chieregatti
E
Gartner
A
Stoffler
HE
Bahler
M
Myr 7 is a novel myosin IX-RhoGAP expressed in rat brain
J Cell Sci
 , 
1998
, vol. 
111
 
Pt 24
(pg. 
3597
-
3608
)
Etienne-Manneville
S
Hall
A
Rho GTPases in cell biology
Nature
 , 
2002
, vol. 
420
 (pg. 
629
-
635
)
Govek
EE
The role of the Rho GTPases in neuronal development
Genes Dev
 , 
2005
, vol. 
19
 (pg. 
1
-
49
)
Hanley
PJ
Xu
Y
Kronlage
M
Grobe
K
Schon
P
Song
J
Sorokin
L
Schwab
A
Bahler
M
Motorized RhoGAP myosin IXb (Myo9b) controls cell shape and motility
Proc Natl Acad Sci U S A
 , 
2010
, vol. 
107
 (pg. 
12145
-
12150
)
Heasman
SJ
Ridley
AJ
Mammalian Rho GTPases: new insights into their functions from in vivo studies
Nat Rev Mol Cell Biol
 , 
2008
, vol. 
9
 (pg. 
690
-
701
)
Impey
S
Davare
M
Lasiek
A
Fortin
D
Ando
H
Varlamova
O
Obrietan
K
Soderling
TR
Goodman
RH
Wayman
GA
An activity-induced microRNA controls dendritic spine formation by regulating Rac1-PAK signaling
Mol Cell Neurosci
 , 
2010
, vol. 
43
 (pg. 
146
-
156
)
Jan
YN
Jan
LY
Dendrites
Genes Dev
 , 
2001
, vol. 
15
 (pg. 
2627
-
2641
)
Kawauchi
D
Taniguchi
H
Watanabe
H
Saito
T
Murakami
F
Direct visualization of nucleogenesis by precerebellar neurons: involvement of ventricle-directed, radial fibre-associated migration
Development
 , 
2006
, vol. 
133
 (pg. 
1113
-
1123
)
Kawauchi
T
Chihama
K
Nabeshima
Y
Hoshino
M
The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration
EMBO J
 , 
2003
, vol. 
22
 (pg. 
4190
-
4201
)
Kobayashi
K
Takahashi
M
Matsushita
N
Miyazaki
J
Koike
M
Yaginuma
H
Osumi
N
Kaibuchi
K
Survival of developing motor neurons mediated by Rho GTPase signaling pathway through Rho-kinase
J Neurosci
 , 
2004
, vol. 
24
 (pg. 
3480
-
3488
)
Koh
C-G
Rho GTPases and their regulators in neuronal functions and development
Neurosignals
 , 
2006
, vol. 
15
 (pg. 
228
-
237
)
Lander
ES
Linton
LM
Birren
B
Nusbaum
C
Zody
MC
Baldwin
J
Devon
K
Dewar
K
Doyle
M
FitzHugh
W
, et al.  . 
Initial sequencing and analysis of the human genome
Nature
 , 
2001
, vol. 
409
 (pg. 
860
-
921
)
Landry
M
Roche
D
Angelova
E
Calas
A
Expression of galanin in hypothalamic magnocellular neurones of lactating rats: co-existence with vasopressin and oxytocin
J Endocrinol
 , 
1997
, vol. 
155
 (pg. 
467
-
481
)
Lee
T
Winter
C
Marticke
SS
Lee
A
Luo
L
Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis
Neuron
 , 
2000
, vol. 
25
 (pg. 
307
-
316
)
McAllister
AK
Cellular and molecular mechanisms of dendrite growth
Cereb Cortex
 , 
2000
, vol. 
10
 (pg. 
963
-
973
)
Moon
SY
Zang
H
Zheng
Y
Characterization of a brain-specific Rho GTPase-activating protein, p200RhoGAP
J Biol Chem
 , 
2003
, vol. 
278
 (pg. 
4151
-
4159
)
Moon
SY
Zheng
Y
Rho GTPase-activating proteins in cell regulation
Trends Cell Biol
 , 
2003
, vol. 
13
 (pg. 
13
-
22
)
Muller
RT
Honnert
U
Reinhard
J
Bahler
M
The rat myosin myr 5 is a GTPase-activating protein for Rho in vivo: essential role of arginine 1695
Mol Biol Cell
 , 
1997
, vol. 
8
 (pg. 
2039
-
2053
)
Nakamura
T
Komiya
M
Sone
K
Hirose
E
Gotoh
N
Morii
H
Ohta
Y
Mori
N
Grit, a GTPase-activating protein for the Rho family, regulates neurite extension through association with the TrkA receptor and N-Shc and CrkL/Crk adapter molecules
Mol Cell Biol
 , 
2002
, vol. 
22
 (pg. 
8721
-
8734
)
Nakayama
AY
Harms
MB
Luo
L
Small GTPases Rac and Rho in the maintance of dendritic spines and branches in hippocampal pyramidal neurons
2000
 
J Neurosci. 20:5329–5338.
Nakazawa
T
Kuriu
T
Tezuka
T
Umemori
H
Okabe
S
Yamamoto
T
Regulation of dendritic spine morphology by an NMDA receptor-associated Rho GTPase-activating protein, p250GAP
J Neurochem
 , 
2008
, vol. 
105
 (pg. 
1384
-
1393
)
Nakazawa
T
Watabe
AM
Tezuka
T
Yoshida
Y
Yokoyama
K
Umemori
H
Inoue
A
Okabe
S
Manabe
T
Yamamoto
T
p250GAP, a novel brain-enriched GTPase-activating protein for Rho family GTPases, is involved in the N-methyl-d-aspartate receptor signaling
Mol Biol Cell
 , 
2003
, vol. 
14
 (pg. 
2921
-
2934
)
Nasu-Nishimura
Y
Hayashi
T
Ohishi
T
Okabe
T
Ohwada
S
Hasegawa
Y
Senda
T
Toyoshima
C
Nakamura
T
Akiyama
T
Role of the Rho GTPase-activating protein RICS in neurite outgrowth
Genes Cells
 , 
2006
, vol. 
11
 (pg. 
607
-
614
)
Newey
SE
Velamoor
V
Govek
EE
Van Aelst
L
Rho GTPases, dendritic structure, and mental retardation
J Neurobiol
 , 
2005
, vol. 
64
 (pg. 
58
-
74
)
Nowak
JM
Grzanka
A
Zuryn
A
Stepien
A
[The Rho protein family and its role in the cellular cytoskeleton]
Postepy Hig Med Dosw (Online)
 , 
2008
, vol. 
62
 (pg. 
110
-
117
)
Okabe
T
Nakamura
T
Nishimura
YN
Kohu
K
Ohwada
S
Morishita
Y
Akiyama
T
RICS, a novel GTPase-activating protein for Cdc42 and Rac1, is involved in the beta-catenin-N-cadherin and N-methyl-D-aspartate receptor signaling
J Biol Chem
 , 
2003
, vol. 
278
 (pg. 
9920
-
9927
)
Post
PL
Bokoch
GM
Mooseker
MS
Human myosin-IXb is a mechanochemically active motor and a GAP for rho
J Cell Sci
 , 
1998
, vol. 
111
 
Pt 7
(pg. 
941
-
950
)
Reczek
D
Bretscher
A
Identification of EPI64, a TBC/rabGAP domain-containing microvillar protein that binds to the first PDZ domain of EBP50 and E3KARP
J Cell Biol
 , 
2001
, vol. 
153
 (pg. 
191
-
206
)
Takei
Y
Teng
J
Harada
A
Hirokawa
N
Defects in axonal elongation and neuronal migration in mice with disrupted tau and map1b genes
J Cell Biol
 , 
2000
, vol. 
150
 (pg. 
989
-
1000
)
Tcherkezian
J
Lamarche-Vane
N
Current knowledge of the large RhoGAP family of proteins
Biol Cell
 , 
2007
, vol. 
99
 (pg. 
67
-
86
)
Van Aelst
L
Cline
HT
Rho GTPases and activity-dependent dendrite development
Curr Opin Neurobiol
 , 
2004
, vol. 
14
 (pg. 
297
-
304
)
Van Aelst
L
D'Souza-Schorey
C
Rho GTPases and signaling networks
Genes Dev
 , 
1997
, vol. 
11
 (pg. 
2295
-
2322
)
Venter
JC
Adams
MD
Myers
EW
Li
PW
Mural
RJ
Sutton
GG
Smith
HO
Yandell
M
Evans
CA
Holt
RA
, et al.  . 
The sequence of the human genome
Science
 , 
2001
, vol. 
291
 (pg. 
1304
-
1351
)
Wang
CL
Zhang
L
Zhou
Y
Zhou
J
Yang
XJ
Duan
SM
Xiong
ZQ
Ding
YQ
Activity-dependent development of callosal projections in the somatosensory cortex
J Neurosci
 , 
2007
, vol. 
27
 (pg. 
11334
-
11342
)
Wong
K
Ren
XR
Huang
YZ
Xie
Y
Liu
G
Saito
H
Tang
H
Wen
L
Brady-Kalnay
SM
Mei
L
, et al.  . 
Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway
Cell
 , 
2001
, vol. 
107
 (pg. 
209
-
221
)
Wong
WT
Faulkner-Jones
BE
Sanes
JR
Wong
RO
Rapid dendritic remodeling in the developing retina: dependence on neurotransmission and reciprocal regulation by Rac and Rho
J Neurosci
 , 
2000
, vol. 
20
 (pg. 
5024
-
5036
)
Zhao
C
Ma
H
Bossy-Wetzel
E
Lipton
SA
Zhang
Z
Feng
GS
GC-GAP, a Rho family GTPase-activating protein that interacts with signaling adapters Gab1 and Gab2
J Biol Chem
 , 
2003
, vol. 
278
 (pg. 
34641
-
34653
)

Author notes

Hui Long and Xinru Zhu contributed equally to this work