Rac signaling impacts a relatively large number of downstream targets; however, few studies have established an association between Rac pathways and pathological conditions. In the present study, we generated mice with double knockout of Rac1 and Rac3 (Atoh1-Cre;Rac1flox/flox;Rac3−/−) in cerebellar granule neurons (CGNs). We observed impaired tangential migration at E16.5, as well as numerous apoptotic CGNs at the deepest layer of the external granule layer (EGL) in the medial cerebellum of Atoh1-Cre;Rac1flox/flox;Rac3−/− mice at P8. Atoh1-Cre;Rac1flox/flox;Rac3−/− CGNs differentiated normally until expression of p27kip1 and NeuN in the deep EGL at P5. Primary CGNs and cerebellar microexplants from Atoh1-Cre;Rac1flox/flox;Rac3−/− mice exhibited impaired neuritogenesis, which was more apparent in Map2-positive dendrites. Such findings suggest that impaired tangential migration and final differentiation of CGNs have resulted in decreased cerebellum size and agenesis of the medial internal granule layer, respectively. Furthermore, Rac depleted/deleted cells exhibited decreased levels of Mid1 and impaired mTORC1 signaling. Mid1 depletion in CGNs produced mild impairments in neuritogenesis and reductions in mTORC1 signaling. Thus, a novel Rac-signaling pathway (Rac1-Mid1-mTORC1) may be involved in medial cerebellar development.

INTRODUCTION

During brain development, migration of immature neurons from germinal zones to their final destination is essential for the establishment of proper circuitry. Two types of neuronal migration – tangential and radial – are required, and radial migration may occur in both inward and outward directions during cerebellar development (Chédotal, 2010; Wang and Zoghbi, 2001). Cerebellar granule neuron (CGN) progenitors are derived at E13-16 from the upper rhombic lip (RL), which is the primary germinal zone of CGNs. Beginning at E13, these progenitor cells migrate tangentially to form the surface of the cerebellar primordium: the external granule layer (EGL), which is the secondary germinal zone of CGNs. After proliferation in the EGL, CGN progenitors begin to differentiate and migrate to deeper layers. Subsequently, CGNs develop inward dendritic processes for radial migration (Kawaji et al., 2004). Inward radial migration, which is completed by P20 in mice (Hatten et al., 1997), is a major characteristic of cerebellar development. By contrast, Purkinje cells (PCs) migrate outwardly from the ventricular zone.

Rac (Rac1-3) is a member of the Rho family of small GTPases and plays important roles in neuritogenesis, including axonogenesis and dendritogenesis, neuronal migration and modulation of gene transcription (Bosco et al., 2009; Funahashi et al., 2014; Govek et al., 2005). Previous studies had shown that, although conditional Rac1 knockout (KO) in telencephalon-derived neurons produced mild impairments in radial migration and axon guidance (Chen et al., 2007; Kassai et al., 2008), these phenotypes were apparently milder than those observed in studies using an electroporated dominant-negative Rac1 mutant (Kawauchi et al., 2003). Rac1-KO mice generated using a Nestin-Cre system exhibited impaired radial migration and axonogenesis in CGNs (Tahirovic et al., 2010). Furthermore, although a relatively large number of downstream molecules and putative disease-related pathways have been suggested to involve Rac signaling, the number of diseases in which involvement of Rac-associated molecules/pathways has been established is quite low (Govek et al., 2005; Stankiewicz and Linseman, 2014).

Midline 1 (MID1) is the gene responsible for Opitz G/BBB syndrome (OS), which is characterized by malformations that affect midline structures, such as laryngotracheoesophageal, cardiac and urogenital abnormalities. Mid1 is a microtubule-associated E3 ubiquitin ligase (Schweiger and Schneider, 2003) that regulates the degradation of the catalytic subunit of protein phosphatase 2A (Ppp2ca) via interaction with the α4 subunit of Pp2a (Trockenbacher et al., 2001). One-third of individuals with OS also exhibit developmental defects of the CNS, which mainly consist of hypoplasia/agenesis of the medial cerebellum (Fontanella et al., 2008; Pinson et al., 2004); however, clinical manifestations of OS in the CNS, as well as in the body, show variable expressivity. Indeed, although Mid1-KO mice have been reported to show hypoplasia of the anterior portion of the medial cerebellum, no other abnormalities were observed (Lancioni et al., 2010). Furthermore, no correlation between the position of the mutation in MID1 and clinical phenotypes of OS has been reported (Fontanella et al., 2008). Thus, the underlying pathogenesis of OS remains to be fully elucidated.

Here, we have investigated the association between downstream targets of Rac and developmental abnormalities using mice with double KO (DKO) of Rac1 and Rac3 in CGNs (Atoh1-Cre;Rac1flox/flox; Rac3−/−). Impaired final differentiation with dendritogenesis of CGNs resulted in agenesis of the medial internal granule layer (IGL) due to disruptions in the radial migration of CGNs. Furthermore, our findings suggest that Mid1 is a novel and transcriptionally regulated Rac target, and that Rac1-Mid1-mTORC1 signaling may be involved in producing agenesis of the medial IGL of Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum.

RESULTS

Rac1 and Rac3 expression in CGNs during development of the IGL

To study the role of Rac GTPases in CGNs, we examined mRNA expression of Rac1 (widely expressed) and Rac3 (neuron specific) in the cerebellum using in situ hybridization. At P6, which is when CGNs begin to migrate radially to form the IGL (Hatten et al., 1997), Rac1 and Rac3 mRNAs were expressed almost exclusively in the EGL (Fig. 1A), where almost all cells are CGNs. At P21, patterns of Rac1 and Rac3 expression became PC dominant, particularly those of Rac3 (Fig. 1B). Slender cells in the molecular layer (ML), which are implicated in the radial migration of CGNs, were Rac1 positive (Fig. S1A). Although a previous study reported an absence of Rac3 expression in the cerebellum (Tahirovic et al., 2010), our results suggested that both Rac1 and Rac3 are expressed in the developing CGNs and that Rac1 expression is more dominant than that of Rac3 (Fig. S1B).

Fig. 1.

Rac1 and Rac3 mRNA expression in CGNs during cerebellar development. (A,B) Sagittal sections of the cerebellum were obtained from P6 (A) and P21 (B) wild-type mice. In situ hybridization of Rac1 (upper panels) and Rac3 (lower panels) was performed using antisense (left panels) and sense (middle panels) DNA probes labeled with digoxigenin-11-dUTP. The right panels show the relative Rac1 and Rac3 mRNA signal levels determined using a digital image analyzer (red was assigned to positive). External granule layer: arrowheads. Purkinje cell layer: arrows. Circles indicate the internal granule layer. Insets are magnified images of the areas indicated by rectangles. Scale bars: 500 µm. n≥4 for each group.

Fig. 1.

Rac1 and Rac3 mRNA expression in CGNs during cerebellar development. (A,B) Sagittal sections of the cerebellum were obtained from P6 (A) and P21 (B) wild-type mice. In situ hybridization of Rac1 (upper panels) and Rac3 (lower panels) was performed using antisense (left panels) and sense (middle panels) DNA probes labeled with digoxigenin-11-dUTP. The right panels show the relative Rac1 and Rac3 mRNA signal levels determined using a digital image analyzer (red was assigned to positive). External granule layer: arrowheads. Purkinje cell layer: arrows. Circles indicate the internal granule layer. Insets are magnified images of the areas indicated by rectangles. Scale bars: 500 µm. n≥4 for each group.

Further deletion of Rac3 in CGNs exacerbates the phenotype of Rac1 KO: agenesis of the IGL in the anterior medial cerebellum of Rac1/Rac3-DKO mice

We generated Rac1-KO mice using Atoh1-Cre mice and Rac1flox/flox mice, hereafter referred to as Atoh1-Cre;Rac1flox/flox mice. Atoh1 is a transcription factor that is essential for the genesis of CGNs (Ben-Arie et al., 1997). Atoh1-positive cells emerge as early as E9.5 in the RL (Akazawa et al., 1995), and Atoh1-positive CGN progenitors migrate from the RL to form the EGL between E12.5 and E16.5 (Wang et al., 2005). The pattern of Atoh1-Cre-driven recombination was assessed using Atoh1-Cre;tdTomato mice. Consistent with the findings of previous reports, tdTomato fluorescence was observed in the developing EGL at the surface of the primordial cerebellum of Atoh1-Cre;tdTomato mice at E16.5, in the EGL at P0, and in both the EGL and IGL at P9. However, no such findings were observed in control mice (CAG-Stopflox-tdTomato) (Fig. 2A). Although Nestin-Cre;Rac1flox/flox mice, in which the Nestin promoter functions in the cerebellum from E15.5 as well as in all neuronal progenitors in the cerebrum around E10.5 (Graus-Porta et al., 2001), were reported to exhibit poor balance (Tahirovic et al., 2010), we detected no obvious behavioral phenotype in Atoh1-Cre;Rac1flox/flox mice. Therefore, we generated DKO mice of Rac1 and Rac3 (Atoh1-Cre;Rac1flox/flox;Rac3−/−) by crossing Atoh1-Cre;Rac1flox/flox mice with Rac3-null mice, which have been reported to show normal microscopic development of the brain (Corbetta et al., 2005). Atoh1-Cre;Rac1flox/flox;Rac3−/− mice exhibited severe ataxic gait when they began walking, and the phenotype was retained throughout life (Movie 1).

Fig. 2.

Loss of the IGL by stepwise genetic deletion of Rac in CGNs using Atoh1-Cre mice. (A) Sagittal sections of the cerebellum were obtained from E16.5, P0 and P9 Atoh1-Cre;tdTomato mice and observed using an LSM 700 (Zeiss). Insets (left, tdTomato fluorescence; right, DAPI) in the E16.5 panel show the negative control obtained from CAG-STOPflox-tdTomato mice. NTZ, nuclear transitory zone; RL, rhombic lip; EGL, external granule layer; IGL, internal granule layer. Scale bars: 500 µm. n≥3. (B) Coronal sections of the cerebellum were obtained from six mouse lines (9 weeks old) and stained using Nissl solution. The rectangle in the low-magnification image of the coronal section (upper left) indicates the location of the cerebellum photographed. Rectangular insets in the lower panels represent magnified images of the regions indicated by yellow rectangles in Atoh1-Cre;Rac1flox/flox (Rac1-KO), Atoh1-Cre;Rac1flox/flox;Rac3−/+ and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3-DKO) cerebella. Arrowheads indicate lightly stained large cells. The thickness of the IGL is indicated by red lines (at the border of agenesis of the IGL in the Rac1/Rac3-DKO cerebellum and the corresponding position in the Rac3-KO (control) and Rac1-KO cerebella) was quantified (data are mean±s.e.m., n≥5, **P<0.01 using Bonferroni's post-hoc test following one-way ANOVA). Scale bars: 300 µm.

Fig. 2.

Loss of the IGL by stepwise genetic deletion of Rac in CGNs using Atoh1-Cre mice. (A) Sagittal sections of the cerebellum were obtained from E16.5, P0 and P9 Atoh1-Cre;tdTomato mice and observed using an LSM 700 (Zeiss). Insets (left, tdTomato fluorescence; right, DAPI) in the E16.5 panel show the negative control obtained from CAG-STOPflox-tdTomato mice. NTZ, nuclear transitory zone; RL, rhombic lip; EGL, external granule layer; IGL, internal granule layer. Scale bars: 500 µm. n≥3. (B) Coronal sections of the cerebellum were obtained from six mouse lines (9 weeks old) and stained using Nissl solution. The rectangle in the low-magnification image of the coronal section (upper left) indicates the location of the cerebellum photographed. Rectangular insets in the lower panels represent magnified images of the regions indicated by yellow rectangles in Atoh1-Cre;Rac1flox/flox (Rac1-KO), Atoh1-Cre;Rac1flox/flox;Rac3−/+ and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3-DKO) cerebella. Arrowheads indicate lightly stained large cells. The thickness of the IGL is indicated by red lines (at the border of agenesis of the IGL in the Rac1/Rac3-DKO cerebellum and the corresponding position in the Rac3-KO (control) and Rac1-KO cerebella) was quantified (data are mean±s.e.m., n≥5, **P<0.01 using Bonferroni's post-hoc test following one-way ANOVA). Scale bars: 300 µm.

Nissl-stained coronal sections of the cerebellum from 9-week-old Atoh1-Cre;Rac1flox/flox;Rac3−/− mice exhibited agenesis of the medial IGL (Fig. 2B). Rac1flox/flox;Rac3−/− (Rac3 KO) mice and Atoh1-Cre;Rac1flox/+;Rac3−/− mice had normal cerebella compared with Atoh1-Cre mice (Fig. 2B). Rac1flox/flox;Rac3−/− mice were subsequently used as controls, in addition to Atoh1-Cre mice and Rac1flox/flox mice. Stepwise exacerbation of hypoplasia of the medial IGL was observed in the cerebellum of Atoh1-Cre;Rac1flox/flox (Rac1 KO) mice and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice following the additional deletion of Rac3 (Fig. 2B). Atoh1-Cre;Rac1flox/flox;Rac3−/+ mice exhibited mild ataxic gait when they began to walk, although the phenotype of these mice was much milder than that of Atoh1-Cre;Rac1flox/flox;Rac3−/− mice throughout life. The thicknesses of the medial IGL in the control, Atoh1-Cre;Rac1flox/flox and Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebella were 143.8±3.1, 40.0±2.2 and 7.5±2.7 µm, respectively (Fig. 2B). In the medial Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum, the ML, PCL and IGL could not be fully distinguished (Fig. 2B). The affected regions (ML, PCL and IGL) of Atoh1-Cre;Rac1flox/flox;Rac3−/+ and Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebella were occupied by multiple layers of lightly stained large cells and densely stained small cells (insets in Figs 2B and 3A,B), whereas Atoh1-Cre;Rac1flox/flox cerebellum exhibited an aligned PCL and thin IGL (insets of Fig. 2B). The lightly stained large cells and some of the densely stained small cells were confirmed as calbindin-positive PCs and tdTomato-positive CGNs, respectively, using 12-week-old Atoh1-Cre;Rac1flox/flox;Rac3−/−;tdTomato mice (Fig. S2).

Fig. 3.

Agenesis of the IGL of the anterior medial cerebellum in Atoh1-Cre;Rac1flox/flox;Rac3−/− mice. (A,B) Rac1flox/flox;Rac3−/− (Rac3 KO=control) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice were fixed at 8 weeks. Sagittal (A) and coronal (B) sections of the cerebellum were stained using Nissl solution. The locations photographed (1-4) are indicated by the lines in the low-magnification coronal section (upper left of A) and sagittal section (upper left of B). I-X in A indicate the lobules of the vermis. Rectangular insets in sagittal and coronal sections (location 1) represent magnified images of the regions indicated by red rectangles in Rac3-KO and Rac1/Rac3-DKO cerebellum. Arrowheads indicate lightly stained large cells. Scale bars: 250 µm in A; 500 µm in B. n≥3.

Fig. 3.

Agenesis of the IGL of the anterior medial cerebellum in Atoh1-Cre;Rac1flox/flox;Rac3−/− mice. (A,B) Rac1flox/flox;Rac3−/− (Rac3 KO=control) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice were fixed at 8 weeks. Sagittal (A) and coronal (B) sections of the cerebellum were stained using Nissl solution. The locations photographed (1-4) are indicated by the lines in the low-magnification coronal section (upper left of A) and sagittal section (upper left of B). I-X in A indicate the lobules of the vermis. Rectangular insets in sagittal and coronal sections (location 1) represent magnified images of the regions indicated by red rectangles in Rac3-KO and Rac1/Rac3-DKO cerebellum. Arrowheads indicate lightly stained large cells. Scale bars: 250 µm in A; 500 µm in B. n≥3.

We then aimed to determine the proportion of the IGL affected by DKO of Rac1 and Rac3 using Nissl-stained serial sagittal (Fig. 3A) and coronal sections (Fig. 3B) of cerebellar tissue from 8-week-old mice. The anterior medial region (anatomically almost identical to the anterior cerebellar lobe) of the IGL (e.g. I-VII folia in the sagittal section) was hypoplastic/aplastic in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum.

Apoptosis of Rac1/Rac3-DKO CGNs in the deep layer of the EGL

We investigated the developmental processes of the EGL and IGL in Atoh1-Cre;Rac1flox/flox;Rac3−/− mice from P4 to P17, when proliferation of CGNs in the EGL and the subsequent formation of the IGL are robust (Hatten et al., 1997). At P4, coronal sections of the cerebellum from Rac1flox/flox;Rac3−/− mice and Atoh1-Cre;Rac1flox/flox;Rac3−/− mice showed comparable EGL thickness and lobular morphology (Fig. 4A). At P7, the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum was apparently smaller than the Rac1flox/flox;Rac3−/− cerebellum (Fig. 4A). Although the thickness of the EGL of the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum increased, the morphology of the EGL became abnormal, particularly in the medial cerebellum (Fig. 4A). At P10, the appearance of the IGL was abruptly absent in the medial Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum, and this phenotype was retained at P17 in combination with the disappearance of the EGL (Fig. 4A).

Fig. 4.

Agenesis of the IGL in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum is caused by apoptosis in the deep layer of the EGL at P8. (A) Age-matched (P4-P17) tissue samples of the cerebellum from Rac1flox/flox;Rac3−/− (Rac3 KO) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice were stained using Nissl solution. Coronal sections of the left Rac1/Rac3-DKO cerebellum show morphological abnormalities in the EGL from P7 and loss of the medial IGL from P10. Scale bars: 300 µm. n≥3. (B) Coronal sections of the left cerebellum at P8 were stained using an active caspase 3 antibody with Hematoxylin counterstaining. Apoptotic cells are detected in the deep layer of the medial EGL of Rac1/Rac3-DKO cerebellum and are indicated by arrowheads in the magnified image (scale bars: 100 µm) of the region indicated by the rectangle (scale bars: 300 µm). n≥3.

Fig. 4.

Agenesis of the IGL in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum is caused by apoptosis in the deep layer of the EGL at P8. (A) Age-matched (P4-P17) tissue samples of the cerebellum from Rac1flox/flox;Rac3−/− (Rac3 KO) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice were stained using Nissl solution. Coronal sections of the left Rac1/Rac3-DKO cerebellum show morphological abnormalities in the EGL from P7 and loss of the medial IGL from P10. Scale bars: 300 µm. n≥3. (B) Coronal sections of the left cerebellum at P8 were stained using an active caspase 3 antibody with Hematoxylin counterstaining. Apoptotic cells are detected in the deep layer of the medial EGL of Rac1/Rac3-DKO cerebellum and are indicated by arrowheads in the magnified image (scale bars: 100 µm) of the region indicated by the rectangle (scale bars: 300 µm). n≥3.

To examine the mechanism of agenesis in the medial IGL of Atoh1-Cre;Rac1flox/flox;Rac3−/− mice between P7 and P10, we used active caspase 3 immunostaining to detect apoptotic cells. Many apoptotic cells were detected in the medial Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum but not in the Rac1flox/flox;Rac3−/− cerebellum at P8 (Fig. 4B), whereas no apoptotic cells were detected in the Atoh1-Rac1flox/flox;Rac3−/− cerebellum at E16.5, P1 or P3. The number of apoptotic cells in the Atoh1-Cre;Rac1flox/flox;Rac3−/+ cerebellum at P8 was ∼50% of that in the Atoh1-Rac1flox/flox;Rac3−/− cerebellum. Moreover, apoptotic cells were accumulated in the deep layer of the medial EGL of Atoh1-Cre;Rac1flox/flox;Rac3−/− mice (Fig. 4B).

To verify the mechanism underlying decreased cerebellum size in Atoh1-Cre;Rac1flox/flox;Rac3−/− mice relative to Rac1flox/flox;Rac3−/− mice, we examined sagittal sections of the cerebellum at E16.5. The distance from the RL to the most anterior point of the EGL, which is the longest path of tangential migration, was analyzed in four sections corresponding to those depicted in Fig. 3A; tangential migrations of Atoh1-Cre;Rac1flox/flox;Rac3−/− CGNs were significantly shorter than those of Rac1flox/flox;Rac3−/− CGNs in all four sections (Fig. 5). However, no significant difference in the thickness of the primordium of the EGL was observed between Atoh1-Cre;Rac1flox/flox;Rac3−/− and Rac1flox/flox;Rac3−/− mice for any of the four sections (Fig. 5). To further examine the proliferative capability of CGNs, we applied EdU pulse labeling to cerebellum at P4, when proliferation of CGNs is high (Hatten et al., 1997). No significant differences in EdU labeling index (ratio of Edu-positive cells/DAPI-positive cells) in the EGL were observed between Atoh1-Cre;Rac1flox/flox;Rac3−/− and Rac1flox/flox;Rac3−/− mice at 4 h after EdU injection (Fig. 6A,B). At 30 h after injection, EdU-labeled CGNs were observed throughout the EGL (Fig. 6A).

Fig. 5.

Impaired tangential migration in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum. Four sagittal sections of the cerebellum were obtained from Rac1flox/flox;Rac3−/− (Rac3 KO) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice at E16.5. The longest distance over which CGN progenitors tangentially migrate from the RL (indicated by the arrow) and the maximum thicknesses of the EGL (bi-directional arrows) were quantified (n=6 for each section from three mice, *P<0.05 using Bonferroni's post-hoc test following two-way ANOVA). Scale bars: 250 µm.

Fig. 5.

Impaired tangential migration in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum. Four sagittal sections of the cerebellum were obtained from Rac1flox/flox;Rac3−/− (Rac3 KO) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice at E16.5. The longest distance over which CGN progenitors tangentially migrate from the RL (indicated by the arrow) and the maximum thicknesses of the EGL (bi-directional arrows) were quantified (n=6 for each section from three mice, *P<0.05 using Bonferroni's post-hoc test following two-way ANOVA). Scale bars: 250 µm.

Fig. 6.

Normal proliferation of CGNs in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum. (A) EdU was injected into Rac1flox/flox;Rac3−/− (Rac3 KO) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice at P4, and samples were fixed at 4 h and 30 h after injection. Sagittal sections of the anterior cerebellar lobe of Rac3-KO and Rac1/Rac3-DKO mice were obtained. Scale bars: 50 µm. (B) EdU labeling index (ratio of Edu-Alexa488-positive cells/DAPI-positive cells) at the EGL 4 h after EdU injection (n=12 from two mice). EGL, external granule layer; ML, molecular layer; IGL, internal granule layer.

Fig. 6.

Normal proliferation of CGNs in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum. (A) EdU was injected into Rac1flox/flox;Rac3−/− (Rac3 KO) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice at P4, and samples were fixed at 4 h and 30 h after injection. Sagittal sections of the anterior cerebellar lobe of Rac3-KO and Rac1/Rac3-DKO mice were obtained. Scale bars: 50 µm. (B) EdU labeling index (ratio of Edu-Alexa488-positive cells/DAPI-positive cells) at the EGL 4 h after EdU injection (n=12 from two mice). EGL, external granule layer; ML, molecular layer; IGL, internal granule layer.

Thus, these results suggested that the smaller size of the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum is due to impaired tangential migration, and that agenesis of the IGL in the medial Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum is caused by apoptosis in the EGL following normal proliferation of CGNs and development of the EGL.

Rac1/Rac3-DKO CGNs differentiate to the final developmental stage

To examine differentiation capabilities of Atoh1-Cre;Rac1flox/flox;Rac3−/− CGNs, we performed immunostaining using: Pax6, a marker for developing CGNs as well as CGNs in the IGL (Yamasaki et al., 2001); p27Kip1, a marker for CGNs located in the deep region of the EGL (Mulherkar et al., 2014; Wang and Zoghbi, 2001); and NeuN, a marker for postmitotic CGNs (Mulherkar et al., 2014) at P5. All CGNs in the EGL were Pax6 positive (Fig. 7A), whereas CGNs in the lower one-third to one half of the EGL were strongly p27Kip1 positive (Fig. 7C). CGNs in the very lowest region (rows 1-3) of the EGL were strongly NeuN positive (Fig. 7D). No significant difference in cerebellar immunostaining for any marker was observed between Atoh1-Cre;Rac1flox/flox;Rac3−/− and Rac1flox/flox;Rac3−/− mice (Fig. 7A-D). The ratio of cell numbers (p27Kip1/DAPI and NeuN/DAPI) in the EGL (statistically analyzed in the anterior and posterior lobe of cerebellum) did not significantly differ between Rac1flox/flox;Rac3−/− and Atoh1-Cre;Rac1flox/flox;Rac3−/− mice (Fig. 7B,C). All lobules (I-X) were observed in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum (Fig. 7B).

Fig. 7.

Normal development of CGNs in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum. (A-D) Rac1flox/flox;Rac3−/− (Rac3 KO) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice were fixed at P5. Sagittal sections of Rac3-KO and Rac1/Rac3-DKO cerebella were stained with a Pax6 antibody (A,B), a p27Kip1 antibody (C) and a NeuN antibody (D). (A,C,D) Magnified images of the anterior (ant) and posterior (post) lobes indicated by rectangles in B are shown. (A) All CGNs in the EGL are Pax6 positive. (B) Lobules I-X are observed in the cerebellum of both Rac3-KO and Rac1/Rac3-KO mice. (C) CGNs in the lower one-third to one-half of the EGL (indicated by yellow lines) are strongly positive for p27Kip1 (n=12 for each region from two mice). (D) CGNs in the lowest region (rows 1-3; indicated by orange lines) of the EGL are strongly positive for NeuN (n=12 for each region from two mice). Scale bars: 25 µm in A,C,D; 500 µm in B. The graphs show the ratio of cell numbers in the EGL (C, p27Kip1/DAPI and D, NeuN/DAPI).

Fig. 7.

Normal development of CGNs in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum. (A-D) Rac1flox/flox;Rac3−/− (Rac3 KO) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice were fixed at P5. Sagittal sections of Rac3-KO and Rac1/Rac3-DKO cerebella were stained with a Pax6 antibody (A,B), a p27Kip1 antibody (C) and a NeuN antibody (D). (A,C,D) Magnified images of the anterior (ant) and posterior (post) lobes indicated by rectangles in B are shown. (A) All CGNs in the EGL are Pax6 positive. (B) Lobules I-X are observed in the cerebellum of both Rac3-KO and Rac1/Rac3-KO mice. (C) CGNs in the lower one-third to one-half of the EGL (indicated by yellow lines) are strongly positive for p27Kip1 (n=12 for each region from two mice). (D) CGNs in the lowest region (rows 1-3; indicated by orange lines) of the EGL are strongly positive for NeuN (n=12 for each region from two mice). Scale bars: 25 µm in A,C,D; 500 µm in B. The graphs show the ratio of cell numbers in the EGL (C, p27Kip1/DAPI and D, NeuN/DAPI).

These results suggest that Atoh1-Cre;Rac1flox/flox;Rac3−/− CGNs undergo apoptosis following differentiation, which appears normal until expression of NeuN occurs.

Requirement of Rac for neuritogenesis and migration of CGNs

To examine neuritogenesis in Rac-deleted CGNs, we used primary cultures of CGNs. Using βIII-tubulin immunostaining, we observed that Atoh1-Cre;Rac1flox/flox CGNs exhibited moderately impaired neuritogenesis compared with Rac1flox/flox and Rac1flox/flox;Rac3−/− CGNs, and that this impairment was exacerbated in Atoh1-Cre;Rac1flox/flox;Rac3−/− CGNs (Fig. 8A).

Fig. 8.

Impaired dendritogenesis in Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellar microexplant and impaired development of vertical neurites of CGNs in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum. (A) After 7 days in culture, primary CGNs from four mouse lines were fixed and stained using a βIII-tubulin antibody and DAPI. The total neurite length was quantified (n=5, **P<0.01 using Bonferroni's post-hoc test following one-way ANOVA). Scale bars: 50 µm. (B,C) After 3 days in culture, cerebellar microexplants from Rac1flox/flox;Rac3−/− (Rac3 KO) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice were fixed and stained using a βIII-tubulin antibody and DAPI (B), or using βIII-tubulin and Map2 antibodies and DAPI (C). (B) The longest neurite lengths indicated by bi-directional arrows (n=17 microexplants from six mice) were quantified (**P<0.01 using the Kolmogorov–Smirnov test). Scale bars: 300 µm. (C) Note the low amount of Map2 immunoreactivity in the image of the Rac1/Rac3-DKO microexplant, which was obtained using the same image acquisition parameters used for imaging of the Rac3-KO microexplant (arrowheads). Scale bars: 100 µm. n=5. (D) Sagittal sections of Atoh1-Cre;tdTomato (control) and Atoh1-Cre;Rac1flox/flox;Rac3−/−;tdTomato (Rac1/Rac3 DKO;tdTomato) mice at P8 were stained using an RFP antibody with DAPI. Arrowheads indicate tdTomato-positive vertical neurites inwardly projecting from radially migrating CGNs in the anterior lobe. The tdTomato-positive vertical neurites are difficult to observe in Rac1/Rac3-DKO mice. EGL, external granule layer; ML, molecular layer; IGL, internal granule layer. Scale bars: 25 µm. n=3.

Fig. 8.

Impaired dendritogenesis in Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellar microexplant and impaired development of vertical neurites of CGNs in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum. (A) After 7 days in culture, primary CGNs from four mouse lines were fixed and stained using a βIII-tubulin antibody and DAPI. The total neurite length was quantified (n=5, **P<0.01 using Bonferroni's post-hoc test following one-way ANOVA). Scale bars: 50 µm. (B,C) After 3 days in culture, cerebellar microexplants from Rac1flox/flox;Rac3−/− (Rac3 KO) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice were fixed and stained using a βIII-tubulin antibody and DAPI (B), or using βIII-tubulin and Map2 antibodies and DAPI (C). (B) The longest neurite lengths indicated by bi-directional arrows (n=17 microexplants from six mice) were quantified (**P<0.01 using the Kolmogorov–Smirnov test). Scale bars: 300 µm. (C) Note the low amount of Map2 immunoreactivity in the image of the Rac1/Rac3-DKO microexplant, which was obtained using the same image acquisition parameters used for imaging of the Rac3-KO microexplant (arrowheads). Scale bars: 100 µm. n=5. (D) Sagittal sections of Atoh1-Cre;tdTomato (control) and Atoh1-Cre;Rac1flox/flox;Rac3−/−;tdTomato (Rac1/Rac3 DKO;tdTomato) mice at P8 were stained using an RFP antibody with DAPI. Arrowheads indicate tdTomato-positive vertical neurites inwardly projecting from radially migrating CGNs in the anterior lobe. The tdTomato-positive vertical neurites are difficult to observe in Rac1/Rac3-DKO mice. EGL, external granule layer; ML, molecular layer; IGL, internal granule layer. Scale bars: 25 µm. n=3.

We then used cerebellar microexplant cultures to further investigate the involvement of Rac in neuritogenesis and migration. Previous research has indicated that, after their final mitosis, CGNs remain in the EGL for 20-48 h. During this period, CGNs change their morphology from round to unipolar to bipolar to tripolar. In the bipolar phase, CGNs reach the deep layer of the EGL. CGNs then inwardly extend a single vertical process and initiate radial migration to form the IGL (Komuro et al., 2001). The horizontal and vertical processes of CGNs are reported to differentiate into the parallel fiber axons and dendrites, respectively (Kawaji et al., 2004). Parallel neurites projecting from the microexplant and neurites perpendicular to the parallel neurites have been reported to mimic the horizontal axons and vertical dendrites of CGNs in vivo, respectively. Biphasic migration, which includes early migration along the parallel neurites and later migration along the perpendicular neurites, observed in the microexplant culture have been used as models of tangential and radial migration in vivo, respectively (Kawaji et al., 2004). The parallel neurite length stained by βIII-tubulin projected from the microexplant (Fig. 8B) and tangential migration, defined as the migration distance along the parallel neurite from the microexplant (Fig. 8B and Fig. S3A), were significantly shorter in Atoh1-Cre;Rac1flox/flox;Rac3−/− microexplants than in Rac1flox/flox;Rac3−/− microexplants. Furthermore, although Rac1flox/flox;Rac3−/− microexplants exhibited regularly aligned parallel neurite bundles in combination with Map2-positive perpendicular neurites, Atoh1-Cre;Rac1flox/flox;Rac3−/− microexplants exhibited impaired alignment of βIII-tubulin-positive neurites with few Map2-positive neurites (Fig. 8C), suggesting severely impaired dendritic development and radial migration in Atoh1-Cre;Rac1flox/flox;Rac3−/− CGNs.

Disrupted vertical neurites in Rac1/Rac3-DKO CGNs in vivo

Next, we examined the vertical neurites projecting from CGNs for radial migration in vivo using Atoh1-Cre;Rac1flox/flox;Rac3−/−;tdTomato mice, in which Rac1/Rac3-DKO CGNs were labeled by tdTomato fluorescence.

Because tdTomato fluorescence in neurites of CGNs was not strong enough for detailed evaluation, and because Map2 immunostaining exhibits a strong non-specific background, tdTomato fluorescence from vertical neurites was enhanced by immunostaining using an RFP polyclonal antibody. Although vertical neurites became visible, horizontal neurites were impossible to evaluate due to the strong tdTomato fluorescence from crowded somata of CGNs in the EGL. Vertical inward neurites projecting from radially migrating CGNs were observed in the Atoh1-Cre;tdTomato cerebellum at P8 (Fig. 8D). In sharp contrast, they were short and poorly visualized in the anterior medial cerebellum of Atoh1-Cre;Rac1flox/flox;Rac3−/−;tdTomato mice (Fig. 8D). However, vertical neurites were visible in the posterior medial Atoh1-Cre;Rac1flox/flox;Rac3−/−;tdTomato cerebellum (Fig. S3B).

Rac enhances the transcription of Mid1

To verify the underlying mechanism of impaired development of the anterior medial cerebellum in Atoh1-Cre;Rac1flox/flox;Rac3−/− mice, we applied DNA microarray analyses to the medial cerebellum at P6, which is just prior to the robust radial migration of CGNs (Hatten et al., 1997) and the emergence of the Atoh1-Cre;Rac1flox/flox;Rac3−/− phenotypes. Among the genes upregulated or downregulated by more than threefold in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum relative to the Rac1flox/flox;Rac3−/− cerebellum, we focused on those encoding cytoskeleton-associated proteins. We revealed that Mid1, which is responsible for abnormalities in midline structures observed in OS, was decreased in the Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebellum compared with control (0.297-fold when normalized using the 75th percentile, 0.289-fold when normalized to Actg1 and 0.297-fold when normalized to Tuba1a). The effect of Rac KO on Mid1 expression detected by DNA microarray analyses was confirmed in the dissected cerebellar cortex, which mainly consists of the EGL, using quantitative PCR (qPCR) (Fig. 9A). Furthermore, we confirmed specific localization of Mid1 mRNA in the EGL as well as decreased Mid1 mRNA in both the EGL of the anterior and posterior lobe of the cerebellum in Atoh1-Cre;Rac1flox/flox;Rac3−/− mice using in situ hybridization (Fig. S4).

Fig. 9.

Mid1 expression is transcriptionally regulated by Rac. (A) Total RNA was isolated from the dissected cerebellar cortex of Rac1flox/flox;Rac3−/− (Rac3 KO) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice at P6. Decreases in levels of Mid1, Rac1 and Rac3 mRNA were quantified using qPCR (n=3, **P<0.01). (B) Forty-eight hours after electroporation of si-Rac1 in RN46A cells, decreases in levels of Mid1 mRNA were quantified using qPCR (n=3, **P<0.01). Knockdown of Rac1 was confirmed via immunoblotting. (C) Twenty-four hours after treatment of RN46A cells with the indicated concentration of a Rac-specific inhibitor, EHop-016. Decreases in levels of Mid1 mRNA were quantified using qPCR (n=6, **P<0.01). (D) Forty-eight hours after mock electroporation or electroporation of HA-Tiam1 into RN46A cells, increases in levels of Mid1 mRNA following HA-Tiam1 transfection were quantified using qPCR (n=6, **P<0.01). (E) Mock, Myc-Rac1 and HA-Tima1 plasmids were transfected into HEK293 cells. Forty-eight hours after transfection, lysates were immunoblotted using Mid1, Myc and HA antibodies. Endogenous Mid1 expression increased slightly following Myc-Rac1 transfection and slightly more following HA-Tiam1 transfection. n=3. Student's t-test and Bonferroni's post-hoc test following one-way ANOVA were applied to A and D, and B and C, respectively.

Fig. 9.

Mid1 expression is transcriptionally regulated by Rac. (A) Total RNA was isolated from the dissected cerebellar cortex of Rac1flox/flox;Rac3−/− (Rac3 KO) and Atoh1-Cre;Rac1flox/flox;Rac3−/− (Rac1/Rac3 DKO) mice at P6. Decreases in levels of Mid1, Rac1 and Rac3 mRNA were quantified using qPCR (n=3, **P<0.01). (B) Forty-eight hours after electroporation of si-Rac1 in RN46A cells, decreases in levels of Mid1 mRNA were quantified using qPCR (n=3, **P<0.01). Knockdown of Rac1 was confirmed via immunoblotting. (C) Twenty-four hours after treatment of RN46A cells with the indicated concentration of a Rac-specific inhibitor, EHop-016. Decreases in levels of Mid1 mRNA were quantified using qPCR (n=6, **P<0.01). (D) Forty-eight hours after mock electroporation or electroporation of HA-Tiam1 into RN46A cells, increases in levels of Mid1 mRNA following HA-Tiam1 transfection were quantified using qPCR (n=6, **P<0.01). (E) Mock, Myc-Rac1 and HA-Tima1 plasmids were transfected into HEK293 cells. Forty-eight hours after transfection, lysates were immunoblotted using Mid1, Myc and HA antibodies. Endogenous Mid1 expression increased slightly following Myc-Rac1 transfection and slightly more following HA-Tiam1 transfection. n=3. Student's t-test and Bonferroni's post-hoc test following one-way ANOVA were applied to A and D, and B and C, respectively.

Because the thickness of the medial IGL in the Atoh1-Cre;Rac1flox/flox cerebellum was 25-30% of that in the control (Rac3-KO) cerebellum (Fig. 2B), we expected that the transcriptional regulation of Mid1 by Rac could be determined by the knockdown (KD) experiment of Rac1 using a validated Rac1-siRNA (Ueyama et al., 2006). A mild decrease in Mid1 mRNA in response to Rac1 KD was observed in rat-derived RN46A neuronal cells using qPCR (Fig. 9B). Transcriptional regulation of Mid1 by Rac was further confirmed by the following results in RN46A cells: decreased Mid1 mRNA levels following treatment with a novel Rac-specific inhibitor (EHop-016; Fig. 9C); and increased Mid1 mRNA levels following expression of a Rac-specific guanine nucleotide-exchange factor (GEF) (Tiam1; Fig. 9D). Increases in Mid1 protein levels following expression of Tiam1 were confirmed in human-derived HEK293 cells (Fig. 9E).

Rac-Mid1-mTORC1 is a novel signal axis involved in the neurite outgrowth of CGNs

Most mammals have a basic pattern of 10 major lobules (I-X) in the cerebellar vermis. We demonstrated that Atoh1-Cre;Rac1flox/flox;Rac3−/− mice exhibited agenesis of the IGL, predominantly in the anterior medial cerebellum. Similarities in abnormal alterations observed in the anterior medial cerebellum of Mid1-KO mice (I-III) (Lancioni et al., 2010) and our Atoh1-Cre;Rac1flox/flox;Rac3−/− mice (I-VII) prompted us to further examine the function and signaling of Mid1 in CGNs. Normal development of the posterior medial cerebellum in both Mid1-KO mice and Atoh1-Cre;Rac1flox/flox;Rac3−/− mice may be explained by the lateral-to-medial movement of CGNs that specifically occurs in the posterior lobe during cerebellar development (Chédotal, 2010; Sgaier et al., 2005).

A recent study demonstrated that inhibition/depletion of Mid1 results in the downregulation of mTORC1 signaling (Liu et al., 2011). This report prompted us to examine a new signaling pathway: Rac1-Mid1-mTORC1. In rat-derived PC12 neuronal cells with Rac1 KD, the protein levels of Ppp2ca, a well-established target of Mid1, were increased relative to control (Fig. 10A). In addition, levels of S6 ribosomal protein and 4E-BP1 phosphorylation, which are well-known downstream targets of mTORC1, were decreased (Fig. 10A). The decreased levels of Mid1 were confirmed using qPCR (Fig. 10A). Decreased levels of Mid1 and mTORC1 signaling (represented by the decreased phosphorylation of 4E-BP1) following Rac deletion were observed in both the medial and lateral cerebellum of Atoh1-Cre;Rac1flox/flox;Rac3−/− mice, using the dissected cerebellar cortex, which mainly consists of the EGL (Fig. 10B). Taken together, these results indicate that Rac is involved in the regulation of the Mid1-mTORC1 signaling axis.

Fig. 10.

The Rac1-Mid1-mTORC1 axis is a novel signaling pathway involved in the neuritogenesis of CGNs. (A) Forty-eight hours after electroporation of the indicated concentration of si-Rac1 in PC12 cells, the levels of Rac1, Ppp2ca, phosphorylated S6 ribosomal protein (Phos-S6) and phosphorylated 4E-BP1 (Phos-4EBP1) were examined via immunoblotting. Decreases in Phos-S6 and Phos-4EBP1, and increases in Ppp2ca levels at 30 nM si-Rac1 were quantified (n=3, *P<0.05 and **P<0.01; Student's t-test). Decreases in the levels of Mid1 mRNA were confirmed using qPCR (n=6, **P<0.01). (B) The levels of Mid1 and Phos-4EBP1 protein were examined in the dissected medial and lateral cerebellar EGLs of Rac1flox/flox;Rac3−/− (Rac3-KO) and Atoh1-Cre;Rac1flox/flox (Rac1/Rac3 DKO) mice at P4 via immunoblotting. Decreases in Phos-4EBP1 and Mid1 levels in the medial and lateral EGLs were quantified (n=3, **P<0.01; Student's t-test). (C) The efficacy of two Mid1 siRNAs (832 and 1107) was confirmed using HEK293 cells with co-transfection of FLAG-tagged mMid1. Forty-eight hours after electroporation of siRNAs (20 nM) in combination with a pEGFP plasmid, mouse primary CGNs were fixed. The longest neurite length of EGFP-positive CGNs was quantified (n=60 from three independent experiments, *P<0.05). The longest neurites were shortened by two si-Mid1s, shown as representative images. Efficient knockdown of Mid1 by two siMid1s in primary CGNs was confirmed using qPCR (n=3, *P<0.05). Scale bars: 25 µm. (D) siRNA (20 nM control or 20 nM siRNA for rat Mid1 [SMARTpool])+pEGFP plasmid was electroporated into primary rat CGNs. The longest neurite length of EGFP-positive CGNs was quantified (n≥60, *P<0.01; Student's t-test). The longest neurites were shortened by si-rMid1. The efficacy of rat Mid1 siRNA was confirmed via qPCR using PC12 cells (n=3, *P<0.05 and **P<0.01). (E) Forty-eight hours after electroporation of the indicated concentration of siRNAs into mouse primary CGNs, levels of Ppp2ca and Phos-S6 were examined via immunoblotting. Decreases in Phos-S6 and increases in Ppp2ca levels at 40 nM siMid1-832 were quantified (n=3, *P<0.05; Student's t-test). Bonferroni's post-hoc test following one-way ANOVA was applied to A-E unless undefined.

Fig. 10.

The Rac1-Mid1-mTORC1 axis is a novel signaling pathway involved in the neuritogenesis of CGNs. (A) Forty-eight hours after electroporation of the indicated concentration of si-Rac1 in PC12 cells, the levels of Rac1, Ppp2ca, phosphorylated S6 ribosomal protein (Phos-S6) and phosphorylated 4E-BP1 (Phos-4EBP1) were examined via immunoblotting. Decreases in Phos-S6 and Phos-4EBP1, and increases in Ppp2ca levels at 30 nM si-Rac1 were quantified (n=3, *P<0.05 and **P<0.01; Student's t-test). Decreases in the levels of Mid1 mRNA were confirmed using qPCR (n=6, **P<0.01). (B) The levels of Mid1 and Phos-4EBP1 protein were examined in the dissected medial and lateral cerebellar EGLs of Rac1flox/flox;Rac3−/− (Rac3-KO) and Atoh1-Cre;Rac1flox/flox (Rac1/Rac3 DKO) mice at P4 via immunoblotting. Decreases in Phos-4EBP1 and Mid1 levels in the medial and lateral EGLs were quantified (n=3, **P<0.01; Student's t-test). (C) The efficacy of two Mid1 siRNAs (832 and 1107) was confirmed using HEK293 cells with co-transfection of FLAG-tagged mMid1. Forty-eight hours after electroporation of siRNAs (20 nM) in combination with a pEGFP plasmid, mouse primary CGNs were fixed. The longest neurite length of EGFP-positive CGNs was quantified (n=60 from three independent experiments, *P<0.05). The longest neurites were shortened by two si-Mid1s, shown as representative images. Efficient knockdown of Mid1 by two siMid1s in primary CGNs was confirmed using qPCR (n=3, *P<0.05). Scale bars: 25 µm. (D) siRNA (20 nM control or 20 nM siRNA for rat Mid1 [SMARTpool])+pEGFP plasmid was electroporated into primary rat CGNs. The longest neurite length of EGFP-positive CGNs was quantified (n≥60, *P<0.01; Student's t-test). The longest neurites were shortened by si-rMid1. The efficacy of rat Mid1 siRNA was confirmed via qPCR using PC12 cells (n=3, *P<0.05 and **P<0.01). (E) Forty-eight hours after electroporation of the indicated concentration of siRNAs into mouse primary CGNs, levels of Ppp2ca and Phos-S6 were examined via immunoblotting. Decreases in Phos-S6 and increases in Ppp2ca levels at 40 nM siMid1-832 were quantified (n=3, *P<0.05; Student's t-test). Bonferroni's post-hoc test following one-way ANOVA was applied to A-E unless undefined.

Finally, we examined the effects of Mid1 KD on neuritogenesis, mTORC1 signaling and migration using mouse primary CGNs and microexplants. Two effective siRNAs for Mid1, siMid1-832 and siMid1-1107 (Fig. 10C) revealed a mild reduction of neuritogenesis (Fig. 10C). The decreased levels of Mid1 were confirmed using qPCR (Fig. 10C). A similar Mid1-KD effect on neuritogenesis was also observed in the experiment using rat primary CGNs and rat-specific siRNA for Mid1 (Fig. 10D). The increased levels of Ppp2ca and the reduced mTORC1 signaling (represented by decreases in the phosphorylation of S6 ribosomal protein) were also confirmed in Mid1-KD (siMid1-832) primary CGNs (Fig. 10E). Furthermore, decreased cell migration was observed in Mid1-KD CGNs of microexplant cultures (Fig. S5).

DISCUSSION

In the present study, we revealed that both Rac1 and Rac3 mRNAs are expressed at the highest levels in CGNs during cerebellar development. Notably, although Rac1 mRNA was still expressed in CGNs at P21, Rac3 mRNA expression disappeared following cerebellar development. Furthermore, the phenotypes of Rac1-KO mice were exacerbated and became tangible by the additional KO of Rac3. These results suggest that both Rac1 and Rac3 function in CGNs during cerebellar development, and that Rac3 is particularly important for CGNs in this period.

Although we demonstrated that both axonogenesis and dendritogenesis, and both tangential and radial migration were impaired in Rac1/Rac3-DKO mice, dendritogenesis and radial migration were more severely impaired both in vitro (cerebellar microexplant) and in vivo (cerebellum). Our findings are in accordance with those of previous reports that have revealed: (1) decreased levels of Map2 and impaired dendrite branching in primary CGNs from Vav3, a GEF for Rac, KO mice (Quevedo et al., 2010); (2) impaired dendritogenesis but not axonogenesis in Rac1-KD primary hippocampal neurons from Rac3-KO mice (Gualdoni et al., 2007); (3) Rac activation by laminin (Gu et al., 2001); and (4) radial migration observed only on laminin-coated dishes in cerebellar microexplant cultures (Kawaji et al., 2004; Nagata and Nakatsuji, 1990).

CGN progenitors migrate tangentially from the RL to form the EGL during the initial stage of cerebellar development (until E16.5) (Sgaier et al., 2005). Rac1-dependency in the tangential migration of olfactory and cortical interneurons has been reported (Chen et al., 2007). In the present study, we demonstrated impaired tangential migration during the formation of the most anterior region of the cerebellar primordium, which requires the longest path of migration, in Rac1/Rac3-DKO mice at E16.5. Because lobules I-X and their EGL were formed at P5, the impaired tangential migration may be related only to the smaller size of the Rac1/Rac3-DKO cerebellum. Impaired differentiation and apoptosis have been reported in Rac1-KO telencephalon-derived neurons (Chen et al., 2009). In addition, selective inhibition of Rac has been reported to induce apoptosis in CGNs (Stankiewicz et al., 2015). Rac1/Rac3-DKO CGNs exhibited: (1) a normal pattern of differentiation until the expression of NeuN, a marker for postmitotic CGNs, was seen in the deepest layer of the EGL at P5; (2) few Map2-positive neurites in cerebellar microexplants and disrupted development of vertical dendrites at P8; and (3) robust apoptosis at the deep layer of the EGL, a premigratory zone for preparing the subsequent radial migration (Yamasaki et al., 2001), at P8. Such results suggest that agenesis of the IGL is due to disruption of the radial migration in CGNs, which is caused by impaired final differentiation of CGNs with dendritogenesis. However, the precise cause of apoptosis in the premigratory zone of the EGL remains unknown.

In the present study, we revealed that Mid1 is a novel and transcriptionally regulated downstream target of Rac. The dynamic phosphorylation/dephosphorylation cycles regulated by Pp2a, a well-established downstream target of Mid1, play important roles in neuronal functions, including migration, neurite initiation and protein synthesis (Ayala et al., 2007; Basu, 2011; Laplante and Sabatini, 2012). Previous studies have reported that 4E-BP1, which is a phosphorylation target of mTORC1 for the promotion of protein synthesis (Nanahoshi et al., 1998), and Map2, which is a well-known dendrite marker involved in neurite outgrowth (Sanchez et al., 2000), are Pp2a targets. Indeed, we observed impairments in mTORC1 signaling and Map2 expression (dendritogenesis) via Rac KD and KO. The protein mTOR assembles into two complexes, mTORC1 and mTORC2, which can be distinguished by their associated proteins, raptor (Rptor) and rictor, respectively (Laplante and Sabatini, 2012). A recent study showed that neuron-specific inactivation of mTORC2 following Rictor KO resulted in a small brain, including a small cerebellum, although the development of the medial IGL was normal (Thomanetz et al., 2013). Meanwhile, another study revealed that neuron-specific inactivation of mTORC1 following Rptor KO resulted in microcephaly and lethality within a few hours of birth, prior to the completion of cerebellar development (Cloetta et al., 2013). These reports exclude the involvement of mTORC2, but not of mTORC1, signaling in CGNs of the medial cerebellum. Rptor-KO mice also exhibit reduced Map2 staining and reduced dendritic complexity in the cerebral cortex (Cloetta et al., 2013). Additionally, Mid1 has been reported to regulate the interaction between mTOR and raptor (Liu et al., 2011), and to regulate microtubule-associated mRNA transport and protein translation (Aranda-Orgillés et al., 2008). Activation of mTORC1 by brain-derived neurotrophic factor (BDNF), which induces Rac activation (Yoshizawa et al., 2005), has been reported to be involved in local protein synthesis in neuronal dendrites (Takei et al., 2004). Impaired protein synthesis in dendrites through impaired Rac-Mid1-mTORC1 signaling may be one of the causes of impaired dendritogenesis and apoptosis of Rac1/Rac3-DKO CGNs in the deep layer, a premigratory zone, of the EGL. However, rescue experiments using GFP-Mid1 into Rac1/Rac3-DKO primary CGNs showed no recovery of impaired neuritogenesis. Although relatively weak involvement of Rac1-mediated transcriptional regulation of Mid1 cannot be completely excluded, these results suggest the following possibilities: (1) in spite of the involvement of Mid1 in neuritogenesis, migration and mTORC1 signaling, Rac is more crucially and broadly involved in these functions than Mid1; and (2) Rac, Mid1 and mTORC1 synergistically act to carry out these functions. Indeed, the extent of cerebellar agenesis in Rac1/Rac3-DKO mice (I-VII) was greater than that of agenesis/hypoplasia reported in the Mid1-KO cerebellum (I-III) (Lancioni et al., 2010). Moreover, we identified the possibility that Rac1/Mid1/mTOR functions as a complex (Fig. S6), in accordance with the findings of previous reports: interactions of Mid1 with mTORC1 (Liu et al., 2011) and Rac1 with mTOR (Saci et al., 2011).

The most frequent CNS phenotype of OS involves agenesis/hypoplasia of the cerebellar vermis, followed by agenesis/hypoplasia of the corpus callosum (Fontanella et al., 2008; Pinson et al., 2004), suggesting that the activity of Mid1 in neurons may be modulated by other factors. Indeed, researchers have revealed agenesis of the vermis (Lancioni et al., 2010) and mild abnormalities in the corpus callosum (Lu et al., 2013) in Mid1-KO mice. In the latter study, elongated axons were observed in Mid1-KD/Mid1-KO cortical neurons (Lu et al., 2013). The cause of the discrepancy between the present study, which revealed impaired neuritogenesis following Mid1 KD, and the latter study remains unknown, although it may be related to differences in the specificity of cortical neurons and CGNs. Alternatively, the activation states of Rac and redundancy of Mid2 (Short et al., 2002) may vary according to the neuronal type/brain region, as well as developmental stage, inducing variable expressivity of OS phenotypes. In any case, phenotypes of Rac1/Rac3-DKO and OS are restricted to the midline. However, we observed that Mid1 and Rac are expressed not only in the medial structures but also the lateral structures/regions (Fig. 10B). Moreover, Mid1 expression and mTORC1 signaling in the Rac1/Rac3-DKO cerebellum was reduced in both the medial and lateral EGL. Such evidence strongly suggests that a specific molecule(s)/signaling(s) exists to regulate the function of Mid1 only around the medial structures of the body. In the present study, we were unable to identify additional factor(s) responsible for the restriction of lesions in OS. However, our findings indicate that Rac1 regulates levels of Mid1 expression and functions synergistically in a complex with Mid1 and mTORC1. A previous study using Nestin-Cre-driven Rac1-KO mice focused on impaired axon formation and neuronal migration in CGNs; however, the authors reported more modest medial cerebellar agenesis (II-V) than that observed in our Rac1/Rac3-DKO mice (I-VII), without referring to any mechanism (Tahirovic et al., 2010). The novel Rac-Mid1-mTORC1 signaling pathway proposed for midline structures in the present study may be further supported by the findings that telencephalon-specific Rac1-KO mice exhibit agenesis of the corpus callosum (Chen et al., 2007; Kassai et al., 2008).

MATERIALS AND METHODS

Animals

Atoh1-Cre transgenic (TG) mice (Matei et al., 2005) and Rac1flox/flox mice (Kassai et al., 2008) were backcrossed to generate Atoh1-Cre;Rac1flox/flox mice. Atoh1-Cre;Rac1flox/flox mice and Rac3−/− mice (Corbetta et al., 2005) were backcrossed for more than five generations to obtain experimental animals (Racflox/flox;Rac3−/−, Atoh1-Cre;Racflox/+;Rac3−/−, Atoh1-Cre;Rac1flox/flox;Rac3−/+ and Atoh1-Cre;Rac1flox/flox;Rac3−/−). CAG-STOPflox-dtTomato (Ai9) TG reporter mice were purchased from the Jackson Laboratory and backcrossed with Atoh1-Cre mice and Atoh1-Cre;Rac1flox/flox;Rac3−/− mice. See the supplementary Materials and Methods for further details of genotyping.

Cells, chemicals, plasmids and RNA interference

RN46A (Sakai et al., 2003), PC12 (Sakai et al., 2003) and HEK293 (ATCC) cells were used in the present study (for details on cell maintenance, see the supplementary Materials and Methods). EHop-016, which inhibits Rac GEF interaction and has 100-fold higher specificity than the established Rac inhibitor NSC23766 (Montalvo-Ortiz et al., 2012), was obtained from Millipore. The details of plasmids and RNA interference are described in the supplementary Materials and Methods.

In situ hybridization

In situ hybridization was performed as previously described (Ueyama et al., 2014) using P6 and P21 wild-type mice and a pair of Rac1flox/flox;Rac3−/− and Atoh1-Cre;Rac1flox/flox;Rac3−/− mice at P5. See the supplementary Materials and Methods for details of DNA probes.

qPCR and RT-PCR

Total RNA was extracted using an RNeasy Mini kit (Qiagen). Reverse transcription was performed on 1-5 µg of total RNA using SuperScript III reverse transcriptase (Invitrogen) and random primers. qPCR was performed using gene-specific primers (Mid1, Rac1, Rac3, Actb and Gapdh) as previously described (Ueyama et al., 2016). Details of primers used for qPCR and RT-PCR can be found in the supplementary Materials and Methods.

Immunoblotting and antibodies

Immunoblotting was performed as previously described (Ueyama et al., 2016). For details of antibodies used in immunoblotting and histochemistry, see the supplementary Materials and Methods.

Section preparation and histochemistry

Sections were prepared as previously described (Ishii et al., 2017). Nissl staining was performed using Cresyl Violet solution (Muto Pure Chemicals). P8 cerebellar sections were used for detecting apoptosis via active caspase 3 immunostaining, followed by diaminobenzidine staining using a Vectastain ABC kit (Vector Laboratories) and Hematoxylin counterstaining. P5 sections obtained from Rac1flox/flox;Rac3−/−;tdTomato and Atoh1-Cre;Rac1flox/flox;Rac3−/−;tdTomato cerebellum were immunostained for Pax6, p27Kip1 and NeuN to evaluate the differentiation of CGNs. P8 sections obtained from Atoh1-Cre;tdTomato and Atoh1-Cre;Rac1flox/flox;Rac3−/−;tdTomato cerebellum were used for immunofluorescence imaging of vertical neurites projecting from radially migrating GCNs. Coronal cerebellar sections obtained from 12-week-old Rac1flox/flox;Rac3−/−;tdTomato and Atoh1-Cre;Rac1flox/flox;Rac3−/−;tdTomato mice were immunostained for calbindin. Samples were imaged using a light microscope with a DP26 camera (Olympus) or a LSM700 confocal laser microscope (Carl Zeiss).

EdU pulse labeling

P4 mice (Atoh1-Cre;Rac1flox/flox;Rac3−/−;tdTomato and Rac1flox/flox;Rac3−/−;tdTomato) were intraperitoneally injected with 15 mg/kg 5-ethynyl-2′-deoxyuridine (EdU) dissolved in PBS. For the analysis of proliferation of CGNs in the EGL, cerebella were fixed 4 h after injection. For the analysis of radial migration of CGNs, cerebella were fixed 30 h after injection. In sagittal sections (8 µm), EdU detection was performed using a Click-iT EdU Alexa Fluor 488 Imaging kit (Invitrogen) and a confocal laser microscope. The EdU labeling index was defined as the ratio of EdU-Alexa488-labeled cells/DAPI-labeled cells.

Preparation of primary CGNs and cerebellar microexplants

Primary CGN cultures and cerebellar microexplant cultures (Nagata and Nakatsuji, 1990) were generated according to a previously described protocol.

For primary CGN cultures, the P8 cerebellum was dissected and dissociated with 0.25% trypsin. Washed and centrifuged cells mixed in DMEM supplemented with B27 (Invitrogen) were plated on poly-D-lysine (PDL)-coated Lab-TekII chamber slides (Nalge Nunc International). In the case of siRNA experiments, 1.0×106 cells were electroporated, and plated on six-well plate dishes (Falcon). CGNs were incubated for the indicated period after addition of Ara-C (final 10 µM) at 24 h.

For cerebellar microexplant cultures, the cerebellum was dissected at P4-P6, and sagittal slices (150 μm) were subsequently prepared with a McIlwain tissue chopper (Mickle Laboratory Engineering). White matter and deep cerebellar nuclei were removed from the tissue slices. Rectangular pieces were then dissected from the remaining tissue, which mainly consisted of the EGL. The prepared rectangular microexplants were placed into DMEM supplemented with B27 on PDL-coated glass-bottomed dishes (MatTek) coated with laminin (Wako) and incubated for 3 days. The rectangular microexplants (from whole, medial and lateral cerebella) were also used for qPCR and immunoblotting analyses.

DNA microarray

Total RNA from the medial region of the Rac1flox/flox;Rac3−/− and Atoh1-Cre;Rac1flox/flox;Rac3−/− cerebella at P6 was extracted using TRIzol (Invitrogen). The quality and quantity of RNA were determined using the Agilent 2100 BioAnalyzer. Gene expression profiles were examined using the SurePrint G3 Mouse Gene Expression 8×60K Microarray kit (Agilent Technologies). The background-subtracted signal intensity of Mid1 was normalized to that of the 75th percentile, Actg1 (γ-actin) or Tuba1a (α-tubulin). The ratios of normalized Mid1 signal (Atoh1-Cre;Rac1flox/flox;Rac3−/−/control) are presented.

Immunoprecipitation

Immunoprecipitation experiments to detect Rac1/MID1/mTOR complex were performed using HEK293 cells. For details, see the supplementary Materials and Methods.

Statistics

All data are presented as the mean±s.e.m. Details of measurements are included in the supplementary Materials and Methods. Two groups were compared using unpaired Student's t-tests or Kolmogorov–Smirnov test. For comparisons of more than two groups, one-way or two-way analyses of variance (ANOVA) were performed, followed by Bonferroni's post-hoc test of pairwise group differences. Statistical analyses were performed using Prism 6.0 software (GraphPad).

Acknowledgements

We thank Prof. Bernd Fritzsch (University of Iowa, USA) for providing Atoh1-Cre TG mice. We also thank Ms Aya Shimizu, MSc and Mr Isao Sakamoto, MPharm for their technical assistance.

Author contributions

Conceptualization: T.U.; Methodology: T.U.; Formal analysis: T.N., T.U., Y.N.; Investigation: T.N., T.U., Y.N., H.S., N.C., Y. Hishikawa, H.K., M.K., E.K., Y. Hisa; Resources: H.K., M.S., I.d.C., A.A.; Data curation: T.U.; Writing - original draft: T.N., T.U.; Writing - review & editing: T.U.; Visualization: T.U.; Supervision: T.U., H.S., E.K., Y. Hisa, N.S.; Project administration: T.U., N.S.; Funding acquisition: T.U., N.S.

Funding

This work was supported by grants from the Japan Society for the Promotion of Science KAKENHI on Innovative Areas ‘Fluorescence Live imaging’ (to N.S.); the Japan Society for the Promotion of Science KAKENHI [17H04042 (to T.U.) and 17024038 (to A.A.)]; the Uehara Memorial Foundation (201320273 to T.U.); and the Hyogo Science and Technology Association (26087 to T.U.).

Data availability

DNA microarray data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-5681.

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Competing interests

The authors declare no competing or financial interests.

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