Arhgef7 is essential for granule cell precursor proliferation and migration during cerebellum development

Vanesa Jiménez-Amilburu; Hugo Ducuing; Nursen Balekoglu; Sneha Sukumaran; Patricia T Yam; Frédéric Charron

doi:10.1016/j.isci.2025.114580

. 2025 Dec 30;29(2):114580. doi: 10.1016/j.isci.2025.114580

Arhgef7 is essential for granule cell precursor proliferation and migration during cerebellum development

Vanesa Jiménez-Amilburu ^1,⁵, Hugo Ducuing ^1,⁵, Nursen Balekoglu ^1,², Sneha Sukumaran ^1,², Patricia T Yam ¹, Frédéric Charron ^1,^2,^3,^4,^6,^∗

PMCID: PMC12828525 PMID: 41585483

Summary

During cerebellar development, granule cell precursors (GCPs) undergo a series of tightly regulated events, including proliferation, migration, and differentiation. Arhgef7, a guanine nucleotide exchange factor for Rac1 and Cdc42, plays a crucial role in these processes. This study investigates the role of Arhgef7 in cerebellar development using conditional knockout (cKO) mice. We demonstrate that Arhgef7 is expressed in GCPs. Loss of Arhgef7 in GCPs results in severe cerebellar hypoplasia and foliation defects, particularly affecting lobules VI/VII. Arhgef7 cKO mice exhibit reduced postnatal GCP proliferation, disorganized cell layers, delayed differentiation, and impaired tangential and radial migration of GCPs. Our findings highlight the essential role of Arhgef7 in regulating multiple aspects of GCP development, thereby ensuring proper cerebellar morphogenesis.

Subject areas: molecular biology, neuroscience, cell biology

Graphical abstract

Highlights

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Arhgef7 is expressed in granule cell precursors (GCPs) during cerebellar development
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Loss of Arhgef7 in GCPs results in severe cerebellar hypoplasia and foliation defects
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Arhgef7 cKO mice exhibit reduced postnatal GCP proliferation and disorganized cell layers
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Arhgef7 cKO mice exhibit impaired migration and potentially delayed differentiation of GCPs

Molecular biology; Neuroscience; Cell biology

Introduction

During the development of the nervous system, a precise sequence of events ensures the formation of functional neuronal circuits. As they are maturing, neurons and their progenitors undergo various phases of proliferation, migration, and differentiation before they start to extend axons and dendrites. Axons and dendrites then navigate toward their target and eventually form synapses with them, thus completing circuit assembly. All these events are tightly orchestrated in time and space by intrinsic genetic programs and locally regulated by extrinsic cues present in the environment of neurons and their progenitors. Though some great advances have been made in recent years, the detailed mechanisms by which such developmental sequences are orchestrated still remain unclear.

In the developing cerebellum, granule cell precursors (GCPs) have been a model of choice to study such developmental sequences. After the first period of proliferation in the upper rhombic lip (RL), GCPs start to leave this territory around embryonic day 12.5 (E12.5) and migrate tangentially on the dorsal surface of the cerebellum to populate and form the external granule layer (EGL).¹^,²^,³ They then undergo a second proliferative phase. Post-mitotic GCPs accumulate under dividing GCPs, thus separating the EGL into a proliferating outer region (oEGL) and a post-mitotic inner region (iEGL). In the iEGL, GCPs can migrate tangentially for a short time using processes parallel to the cerebellum surface and start to express differentiation markers. They then switch their migration mode from tangential to radial. As a result, they extend a third, perpendicular process, in the molecular layer (ML) and toward the internal granule layer (IGL) underneath, and the two other parallel processes grow to become parallel fibers. GCPs then migrate radially by nucleokinesis along their perpendicular process, through the ML, until they reach their final location in the IGL and finish their differentiation into cerebellar granule neurons (CGNs).¹^,²^,³

These events are tightly regulated by a multitude of intrinsic and extrinsic factors. The early specification of GCPs in the RL is controlled by transcription factors, such as Math1,⁴^,⁵^,⁶ and secreted cues, such as bone morphogenetic protein and fibroblast growth factors.⁷^,⁸^,⁹ The proliferation in the oEGL is mainly regulated by Sonic hedgehog (Shh) secreted by Purkinje cells.¹⁰^,¹¹^,¹² The migration of GCPs is thought to be mainly controlled by extracellular cues, such as Slit and CXCL12 during the initial tangential migration¹³^,¹⁴ and BDNF, Sema6A, and Netrin-1 during the radial migration.¹⁵^,¹⁶^,¹⁷ Notably, in vitro data suggest that the radial migration of GCPs is partly an intrinsic property that does not require external signals.¹⁸

Along their journey through this myriad of cues, the question remains as to how GCPs integrate them precisely during the development of the cerebellum. Small Rho GTPases are crucial signaling components that could be good candidates. In particular, Rac1 and Cdc42 play a variety of roles during the development of the nervous system, and in particular during the development of the cerebellum.¹⁹^,²⁰ Notably, these small Rho GTPases work in tandem with their activators, the guanine nucleotide exchange factors (RhoGEF). Arhgef7 (also known as β-PIX) is a RhoGEF for Rac1 and Cdc42.²¹^,²² Arhgef7 interacts with the GIT proteins, GIT1 and GIT2,²³^,²⁴ and together they form regulatory complexes that function in many cellular processes, including cell migration.²² Although various studies have shown that Arhgef7 is important during neurodevelopment,²⁵^,²⁶^,²⁷^,²⁸^,²⁹^,³⁰^,³¹ its role in the development of GCPs has never been investigated.

In this study, we show that loss of Arhgef7 in mouse GCPs led to hypoplasia and foliation defects in the cerebellum. We also observed that the loss of Arhgef7 impaired GCP proliferation, intercellular organization, differentiation, and tangential and radial migrations. Overall, our study highlights the importance of Arhgef7 in cerebellar development.

Results

Arhgef7 is expressed by GCPs during cerebellar development

To determine whether Arhgef7 RNA is expressed in developing GCPs of the cerebellum, we used a single cell RNA sequencing (scRNA-seq) dataset of embryonic and postnatal (from E10 to P14) cerebellum development.³² After removing blood and vascular cells, we were left with 57,928 cells (Figure 1A). Two distinct clusters of GCPs were visible: proliferating GCPs (highly positive for Ki67; Figure 1B) and post-mitotic GCPs (highly positive for Tag-1; Figure 1C). Arhgef7 is expressed in both clusters, with higher levels in the post-mitotic GCP cluster (Figure 1D). Arhgef7 is also expressed in other cells of the cerebellum, such as Purkinje cells and Bergmann glia.

We next assessed Arhgef7 at the protein level, at two time points of GCP development. We used an Arhgef7 antibody that has been previously validated in germline knockout tissue and used for immunostaining.³¹ Immunofluorescence staining of sagittal cerebellar sections at E17.5 showed that proliferative GCPs express Arhgef7 in the EGL (Figure 1E). Consistent with the scRNA-seq data, other cell types of the developing cerebellum also express Arhgef7. At P7, GCPs (and other cell types) continue to express Arhgef7 (Figure 1F). In the P7 EGL, higher levels of Arhgef7 protein were observed in the iEGL (post-mitotic GCPs) compared with the oEGL (proliferating GCPs), mirroring what we observed at the RNA level by scRNA-seq. Thus, our results show that GCPs, along with other cerebellar cell types, express Arhgef7 RNA and protein during cerebellar development.

Arhgef7 cKO mice display severe hypoplasia and morphogenesis defects in the cerebellum

Arhgef7^−/− mice die embryonically at E8.5, prior to cerebellum formation.³³ Thus, to investigate the role of Arhgef7 in GCP development, we generated Arhgef7 conditional knockout (cKO) mice. Math1-Cre is expressed in GCPs starting around E12.5.⁵^,⁶^,³⁴ Therefore, we used Math1-Cre to conditionally remove Arhgef7 from GCPs. Arhgef7 conditional knockouts (Arhgef7 cKO) were Math1-Cre⁺;Arhgef7^fl/- and control animals were Math1-Cre^-;Arhgef7^+/fl. We confirmed that conditional removal of Arhgef7 cKO leads to a decrease in Arhgef7 protein levels in the EGL at E17.5 and P7 (Figures S1A and S1B). Consistently, we also observe a decrease in Arhgef7 in the IGL at P7. Of note, this partial reduction is due to the fact that Arhgef7 cKO animals still express a truncated form of the Arhgef7 protein after recombination of loxP sites.³³ Although this truncated form of Arhgef7 is not functional, it is still detected by the anti-Arhgef7 antibody.

Arhgef7 cKO mice are viable and grow to adulthood. To investigate the impact of Arhgef7 deletion in GCPs on cerebellar development, we first analyzed the cerebella of control and Arghef7 cKO mice at P60. Arhgef7 cKO cerebella displayed severe hypoplasia compared with controls (Figure 2A). We then stained sagittal sections of these cerebella with DAPI, allowing us to visualize the morphology and organization of the lobules and cell layers (Figure 2A). Arhgef7 cKO cerebella had a ∼50% reduction in the total area of the cerebellum (Figure 2B). In the IGL, where all GCPs have migrated and differentiated at this stage, Arhgef7 cKO mice had a ∼55% reduction in the area (Figure 2C). In addition, the morphology of many lobules was affected in Arhgef7 cKO cerebella, notably showing less elongated lobules (Figure 2A). However, not all lobules were equally affected, ranging from a 19% decrease in area in lobule X to a 64% decrease in lobule VI/VII, and from a 10% decrease in white matter length in lobule X to a 67% decrease in lobule VI/VII (Figures S2D and S2E). Lobule VI/VII was the most affected, with a complete lack of the intercrural fissure between lobule VIb and lobule VII.

*Arhgef7* cKO mice display severe hypoplasia and morphogenesis defects in the cerebellum

(A) Pictures of P60 control and *Arhgef7* cKO cerebella (outlined in red) and images of sagittal sections of P60 control and *Arhgef7* cKO cerebella stained with DAPI. The numbering of each lobule is indicated. The intercrural fissure is indicated by a white arrow. Scale bar: 1 mm.

(B and C) Quantifications of cerebellum area (B) and IGL area (C) in medial sagittal sections of P60 control and *Arhgef7* cKO mice. Data are represented as mean ± SEM. Number of animals: control n = 4, *Arhgef7* cKO n = 3. Unpaired t test; ∗∗∗∗p < 0.0001.

(D) Images of sagittal sections of E17.5 control and *Arhgef7* cKO cerebella stained with DAPI. Early anchoring points are indicated by yellow arrowheads. Scale bar: 200 μm.

(E and F) Quantifications of cerebellum area (E) and EGL area (F) in medial sagittal sections of E17.5 control and *Arhgef7* cKO mice. Data are represented as mean ± SEM. Number of animals: control n = 4, *Arhgef7* cKO n = 4. Unpaired t test; ns, not significant.

(G) Images of sagittal sections of P7 control and *Arhgef7* cKO cerebella stained with DAPI. Early anchoring points are indicated by yellow arrowheads. Images on the second row show higher magnification of the yellow boxed areas in the images on the first row. The uvular sulcus in lobule IX is indicated by a white arrowhead. Anchoring points in lobule VI/VII are indicated by yellow arrowheads. Scale bar: 200 μm.

(H–K) Quantifications of cerebellum area (H), EGL area (I), IGL area (J), and ML thickness (K) in medial sagittal sections of P7 control and *Arhgef7* cKO mice.

Data are represented as mean ± SEM. Number of animals: control n = 4, *Arhgef7* cKO n = 3. Unpaired t test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

We next analyzed the timing of the onset of these defects during development. To do so, we dissected cerebella from E15.5, E17.5, and P7 animals and stained sagittal sections of these cerebella with DAPI. At E15.5, Arhgef7 cKO cerebella showed no defect in whole cerebellum area, morphology, or cell layer organization (Figures S2A–S2C). At E17.5, Arhgef7 cKO cerebella showed no significant difference in cerebellum area and EGL area (Figures 2D–2F). Interestingly, E17.5 Arhgef7 cKO appear to be missing the early anchoring centers that will lead to the formation of the four cardinal lobes (Figure 2D, yellow arrowheads). The establishment of these anchoring centers is unlikely to be lost but rather only delayed, as the cardinal lobes are eventually formed at later stages in Arhgef7 cKO cerebella. At P7, Arhgef7 cKO displayed cerebellar hypoplasia (Figure 2G), with a ∼30% decrease in the cerebellum area (Figure 2H), which was also reflected strongly in the EGL and the IGL areas (Figures 2I and 2J) and to a lesser extent in the ML thickness (Figure 2K). Similarly to P60 cerebella, not all lobules were equally affected at P7, ranging from a 4% decrease in area in lobule X to a 52% decrease in lobule VI/VII (Figure S2F). Again, lobule VI/VII was the most affected. Notably, in addition to the medial cerebellum sections (which we described thus far), this hypoplasia was also observed in lateral sections (Figures S2G–S2J).

We also found that the morphology of P7 Arhgef7 cKO cerebella is affected in other ways. Generally, outer convex regions were more rounded compared with controls. More strikingly, the uvular sulcus in lobule IX was absent in Arhgef7 cKO (Figure 2G, white arrowhead), as well as the anchoring centers that will form the sulcus between lobules VIa and VIb and the fissure between lobules VI and VII (Figure 2G, yellow arrowheads). By comparing with P60 animals, we can infer that the uvular sulcus formation is delayed but that the anchoring points between lobules VIa, VIb, and VII never fully develop. Overall, our results show that Arhgef7 is required in GCPs for normal cerebellar development and that the loss of Arhgef7 impairs morphogenesis of the cerebellum. Also, we observed none of the aforementioned morphological defects in P7 sagittal sections of germline heterozygous mutant animals (Math1-Cre^-;Arhgef7^fl/-) or conditional heterozygous mutant animals (Math1-Cre⁺;Arhgef7^+/fl) (Figures S2K–S2M). This indicates that the loss of one copy of Arhgef7 in the whole animal or in GCPs is not enough to cause defects in cerebellar development.

Arhgef7 cKO mice have a disorganized EGL architecture

To further characterize the phenotype of Arhgef7 cKO cerebella, we investigated the organization of cell layers in the cerebellum in sagittal sections stained with either DAPI or hematoxylin and eosin (H&E). At E17.5, we observed a strong disorganization at the border between the EGL and the ML in Arhgef7 cKO cerebella, which appeared rough instead of smooth and straight (Figure 3A). Both the EGL and the ML thicknesses in Arhgef7 cKO were very variable. This disorganization remained present in P7 Arhgef7 cKO cerebella but was mainly localized to the primary fissure at that stage, between lobule V and VI (Figures 3B and 3G). Again, the border between the EGL and the ML was rough and the thicknesses of the EGL and the ML was very variable in Arhgef7 cKO cerebella. We quantified this disorganization by measuring the thickness of the EGL at the entrance of the fissure, at the bottom, and midway in-between, on the posterior side of the fissure (Figure 3B, red bars). Though control and Arhgef7 cKO EGL had the same average thickness (Figure 3C), the range of thickness measurements were over three times larger in Arhgef7 cKO mice compared with control mice, illustrating the severe disorganization of the EGL at the border with the ML (Figure 3D). This effect was lobule specific as lobule IX did not show this defect (Figures 3E and 3F).

Since we observed a disorganization at the border between the EGL and the ML, we also stained P7 control and Arhgef7 cKO sagittal cerebellar sections for Calbindin, a marker that labels Purkinje cells in the ML, and counterstained with DAPI (Figure 3G). We did not observe any drastic difference in the architecture of the ML, the density of Purkinje cells, or their morphology. In both conditions, Purkinje cells dendritic trees were developed, spawning from the cell body close to the IGL and reaching the border of the iEGL. The dendritic trees did not invade the EGL in Arhgef7 cKO cerebella. This suggests that the GCPs are responsible for the disorganization at the border between the EGL and the ML.

Tag-1 is a marker of post-mitotic, differentiating GCPs in the iEGL. Immunohistochemical stainings against Tag-1 showed that the border between the oEGL and the iEGL was unaffected in Arhgef7 cKO, and that the disorganized GCPs at the border between the iEGL and the ML are Tag-1 positive (Figures S3A and S3B). This suggests that one aspect of the disorganization is caused by differentiating GCPs in the iEGL. In addition, H&E staining imaged at high magnification revealed further morphological defects in the oEGL of Arhgef7 cKO cerebella (Figure 3H). In control cerebella, GCP nuclei in the oEGL were often larger and more diffusely stained (Figure 3H, red arrow) compared with GCP nuclei in the iEGL, which were smaller and more densely stained (Figure 3H, red asterisk). In Arhgef7 cKO cerebella, this distinction was not as clear, as we observed small and more densely stained GCP nuclei in the oEGL (Figure 3H, red asterisk), as well as larger and more diffusely stained GCP nuclei deeper in the iEGL (Figure 3H, red arrow). Similarly to the hypoplasia, this aspect of the disorganization within the oEGL/iEGL displayed lobule-dependent variability. For instance, the preculminate fissure (between lobules III and IV/V) and the primary fissure (between lobules IV/V and VI/VII) were much more affected than the secondary fissure (between lobules VIII and IX) (Figure S3C). The intermingling of smaller and larger nuclei we observed with the H&E staining does not reflect an intermingling of proliferating and differentiating states, as shown by Tag-1 immunostainings (Figure S3B). Rather, the size of the nuclei might reflect chromatin condensation states, which is likely to be independent of the proliferation vs. differentiation status of GCPs. In P60 animals, when the EGL has disappeared and GCPs have migrated to the IGL, no disorganization was observed in the IGL of Arhgef7 cKO cerebella (Figure S3D).

Overall, our results show that the loss of Arhgef7 disorganizes the architecture of the EGL both i) within the iEGL and oEGL and also ii) at the border between the iEGL and the ML, where the differentiating, pre-migratory GCPs are located.

Loss of Arhgef7 might disrupt embryonic tangential migration of GCPs

We next investigated the impact of the loss of Arhgef7 in the migration of GCPs. GCP tangential migration starts around E13 as GCPs exit the RL and migrate tangentially on the cerebellum surface to form the EGL and ends around E18.5. To evaluate this tangential migration, we measured the length of the EGL in sagittal sections of E17.5 control and Arhgef7 cKO cerebella stained with DAPI (Figure 4A, red dashed line). To minimize medio-lateral variability, we quantified only the most medial sections of each embryo. Although the total perimeter of the cerebellum was unchanged in Arhgef7 cKO mice (Figure 4B), the length of the EGL was 15% shorter in Arhgef7 cKO mice (Figure 4C) and only represents 48% of the cerebellar perimeter compared with 60% in the control (Figure 4D). This suggests that tangential migration might be impaired following the loss of Arhgef7 in GCPs.

Loss of *Arhgef7* disrupts embryonic tangential migration and postnatal radial migration of GCPs

(A) Images of sagittal sections of E17.5 control and *Arhgef7* cKO cerebella stained with DAPI. The measured length of the EGL is indicated by a red dotted line. RL, rhombic lip. Scale bar: 100 μm.

(B–D) Quantifications of the cerebellum perimeter (B), the EGL length (C), and the ratio between the EGL length and the cerebellum perimeter (D) in sagittal sections of E17.5 control and *Arhgef7* cKO cerebella. Data are represented as mean ± SEM. Number of animals: control n = 3, *Arhgef7* cKO n = 3. Unpaired t test; ∗p < 0.05; ns, not significant.

(E) Images of sagittal sections of P7 control and *Arhgef7* cKO cerebella stained with DAPI. Examples of GCPs presenting an elongated nucleus are indicated by yellow arrows. Scale bar: 50 μm.

(F and G) Immunostaining of sagittal sections of P9 control and *Arhgef7* cKO cerebella stained for EdU (green) and counterstained with DAPI (magenta). Control and *Arhgef7* cKO animals received one injection of EdU at P7. Images show the whole cerebellum (F), or higher magnifications of the primary fissure area of lobule VI, the precentral fissure area of lobule III, and the uvular fissure area of lobule IX (G). Lobules III, VI, and IX are indicated. Scale bars: 500 μm in (F) and 50 μm in (G).

(H–J) Quantification of the density of EdU⁺ cells and the repartition of these cells within the cerebellum layers in the primary fissure area of lobule VI (H), the precentral fissure area of lobule III (I), and the uvular fissure area of lobule IX (J). EGL, external granule layer; ML, molecular layer; IGL, internal granule layer. Data are represented as mean ± SEM. Number of animals: control n = 4, *Arhgef7* cKO n = 4. Unpaired t test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001; ns, not significant.

Loss of Arhgef7 impairs postnatal radial migration of GCPs

We next examined the effect of the loss of Arhgef7 in GCPs during radial migration, which starts after birth (∼P3). Differentiating GCPs in the iEGL migrate perpendicularly to the cerebellum surface through the ML to eventually settle in the IGL. This radial migration peaks around P9 and ends around P20. At P7, in control cerebella, we observed numerous GCP nuclei inside the ML with an elongated shape along an axis perpendicular to the cerebellar surface (Figure 4E, yellow arrows). This is typical of GCPs migrating radially along Bergmann glial fibers through nucleokinesis.²⁰^,³⁵^,³⁶^,³⁷^,³⁸ In Arhgef7 cKO cerebella, however, in the primary fissure region of lobule VI, we observed far fewer GCP nuclei with this elongated shape. This suggests a defect in radial migration due to the loss of Arhgef7 in GCPs.

To evaluate more directly radial migration, we performed EdU tracing experiments. We injected P7 pups with EdU, harvested their cerebella at P9, and stained their sagittal sections with DAPI (Figure 4F). We quantified the proportion of EdU⁺ cells in each layer in which post-mitotic GCPs can be found, i.e., the EGL, the ML, and the IGL. Interestingly, in the primary fissure in lobule VI, we found more EdU⁺ GCPs in the EGL and the ML and fewer EdU⁺ GCPs in the IGL of Arhgef7 cKO cerebella compared with controls (Figures 4G and 4H). This indicates that the loss of Arhgef7 in GCPs hinders their radial migration. Additionally, it suggests that their migration is slower, as fewer GCPs have reached the IGL after 2 days, with many still remaining in the EGL and ML. This impairment showed regional difference throughout the cerebellum. Indeed, in the precentral fissure in lobule III, we observed a small increase in EdU⁺ GCPs inside the EGL, no difference in the ML, and a small reduction in the IGL of Arhgef7 cKO cerebella compared with controls (Figures 4G and 4I). In contrast, we observed no difference in all layers in the uvular fissure in lobule IX (Figures 4G and 4J). In the whole lobule VI/VII, we observed the same trend (although not significant) as in our initial quantification at the primary fissure: a slight increase in EdU⁺ in the EGL of Arhgef7 cKO cerebella compared with controls and reflected by a slight decrease in the IGL (Figure S4A). However, we did not observe such trend at the intercrural fissure (which is the fissure that eventually separates lobules VI and VII in the controls and which is not generated in Arhgef7 cKO animals; Figure 2A), suggesting that the loss of Arhgef7 does not affect the migration of GCPs in this region at this stage (Figure S4B). Importantly, our H&E stainings at P60 show that, following the loss of Arhgef7, despite a cerebellum hypoplasia, GCPs are still able to eventually complete their migration correctly and settle in the IGL (Figure S3B).

Arhgef7 cKO cerebella display reduced postnatal proliferation, potentially delayed differentiation, and no increase in apoptosis

Since Arhgef7 cKO cerebella display severe hypoplasia, we next investigated cell death, cell proliferation, and cell differentiation during cerebellar development.

To investigate cell death, we performed Caspase3 stainings on sagittal sections of P7 control and Arhgef7 cKO cerebella (Figure 5A). We detected very few Caspase3⁺ cells in control and Arhgef7 cKO cerebella compared with our positive control (a Ptch1^+/− cerebellar tumor³⁹) (Figure 5A), and there was no significant difference in the density of Caspase3⁺ cells between control and Arhgef7 cKO in the EGL, ML, and IGL (Figure 5B). Thus, Arhgef7 cKO in GCPs does not impact apoptosis during cerebellar development.

*Arhgef7* cKO cerebella display reduced postnatal proliferation, potentially delayed differentiation, and no increase in apoptosis

(A) Immunostaining of sagittal sections of P7 control and *Arhgef7* cKO cerebella stained for Caspase3 (green) and counterstained with DAPI (magenta). Cerebellar tumor was used as a positive control. Scale bar: 50 μm.

(B) Quantification of the density of Caspase3⁺ cells in the primary fissure area of lobule VI of P7 control and *Arhgef7* cKO cerebella. EGL, external granule layer; ML, molecular layer; IGL, internal granule layer. Data are represented as mean ± SEM. Number of animals: control n = 2, *Arhgef7* cKO n = 2.

(C) Immunostaining of sagittal sections of E17.5 control and *Arhgef7* cKO cerebella stained for Ki67 (green) and counterstained with DAPI (magenta). Scale bar: 100 μm.

(D) Quantification of the density of Ki67⁺ cells in the EGL of sagittal sections of E17.5 control and *Arhgef7* cKO cerebella. Data are represented as mean ± SEM. Number of animals: control n = 3, *Arhgef7* cKO n = 3. Unpaired t test; ns, not significant.

(E) Immunostaining of sagittal sections of P7 control and *Arhgef7* cKO cerebella stained for Ki67 (green) and NeuN (cyan) and counterstained with DAPI (magenta). EGL, external granule layer; ML, molecular layer; IGL, internal granule layer. Scale bars: 100 μm.

(F and G) Quantification of the percentage of Ki67⁺ and NeuN⁺ over DAPI⁺ cells in the EGL of lobule VI at the primary fissure of sagittal sections of P7 control and *Arhgef7* cKO cerebella. Data are represented as mean ± SEM. Number of animals: control n = 4, *Arhgef7* cKO n = 3. Unpaired t test; ∗∗p < 0.01.

(H) Quantification of the percentage of NeuN⁺ over DAPI⁺ cells in the EGL, ML, and IGL of lobule VI at the primary fissure of sagittal sections of P7 control and *Arhgef7* cKO cerebella.

Data are represented as mean ± SEM. Number of animals: control n = 4, *Arhgef7* cKO n = 3. Unpaired t test; ∗p < 0.05; ns, not significant.

We next investigated proliferation. We stained sagittal cerebellar sections at E17.5 and at P7 using the proliferation marker Ki67 (Figures 5C and 5E). At both stages and for both controls and Arhgef7 cKO, the vast majority of Ki67⁺ cells we observed were located in the oEGL, where proliferating GCPs normally reside. This suggests that loss of Arhgef7 does not impair the compartmentalization of proliferating and differentiating GCPs during cerebellar development, but rather mainly the border between the iEGL and the ML (Figures 5C and 5E). At E17.5, the number of Ki67⁺ cells was not changed in Arhgef7 cKO cerebella compared with control (Figure 5D), suggesting that loss of Arhgef7 does not affect proliferation at this stage. This is consistent with the fact that the cerebellum and EGL area are not significantly different between Arhgef7 cKOs and controls at this stage (Figures 2D–2F). At P7, we observed a decrease (from 61.6% to 55.3%) in the percentage of Ki67⁺ cells (over DAPI) in the EGL of Arhgef7 cKO cerebella compared with control (Figure 5F). We also stained for the mitosis marker pH3 and, consistent with the Ki67 results, we observed fewer pH3⁺ GCPs in Arhgef7 cKO than in control cerebella (Figures S5A and S5B). Interestingly, this effect was only localized to some areas within the cerebellum; we observed no difference in the density of pH3⁺ cells in the whole lobule VI/VII (Figure S5C). In contrast, there was a ∼20% decrease in pH3⁺ GCPs in the primary fissure between lobule V and VI of Arhgef7 cKO cerebella (Figure S5B). Notably, this is the region where the iEGL/ML border disorganization was the most severe (Figures 3B–3D).

The decrease in the percentage of EGL GCPs that are Ki67⁺ (Figure 5F) might be due to an increase in the proportion of differentiated cells. While we did not see major differences in differentiation using Tag-1 (Figures S3A and S3B), we next used a marker that allows a more quantitative assessment of differentiation. For this, we stained for the differentiation marker NeuN, a marker that strongly labels post-mitotic CGNs in the IGL and faintly labels differentiating, pre-migratory GCPs at the border of the iEGL (Figure 5E). We observed an increase (from 38.4% to 44.7%) in the percentage of NeuN⁺ cells in the EGL of Arhgef7 cKO cerebella compared with control (Figure 5G). Although this initially suggested that Arhgef7 might influence the balance between GCP proliferation and differentiation, a different picture emerged when, in addition to the EGL, we also analyzed the ML and IGL. When we counted the percentage of NeuN⁺ cells in the EGL (over the number of DAPI cells in the EGL+ML+IGL), we saw an increase from 16.9% to 22.2% (a difference of 5.3%; Figure 5H) in Arhgef7 cKOs compared with controls. In contrast, when we quantified the percentage of NeuN⁺ cells in the ML + IGL (over the number of DAPI cells in the EGL+ML+IGL), we observed a decrease (from 56.1% to 50.2%; a difference of 5.9%), similar to the increase in the EGL (Figure 5H). Consistently, when we measured the percentage of NeuN⁺ cells in the EGL+ML+IGL, we did not see a difference between Arhgef7 cKOs and controls (Figure 5H). Thus, the proportion of NeuN⁺ cells in Arhgef7 cKOs vs. controls is not significantly changed, suggesting that there does not seem to be a major impact of inactivating Arhgef7 on differentiation. Interestingly, what appears to be changing is the layer distribution of NeuN⁺ cells, where we see more NeuN⁺ cells in the EGL of the Arhgef7 cKOs compared with controls (Figures 5G and 5H). This might be due to more NeuN⁺ cells temporarily lingering in the EGL of Arhgef7 cKOs due to a migration defect. Alternatively, loss of Arhgef7 might slow down the differentiation of GCPs, leading to their accumulation within the iEGL until they finish differentiation and eventually migrate to the ML and IGL. Both possibilities are consistent with our EdU pulse-chase experiments (Figures 4F–4H).

We next investigated in more detail the potential causes underlying the decrease in Ki67⁺ cells in the Arhgef7 mutant EGL (Figures 5E and 5F). For this, we looked at the EdU⁺/Ki67⁺ cell fraction in our animals injected with EdU at P7 and harvested 48h later. Using EdU and Ki67 immunostaining, we quantified the percentage of EdU⁺;Ki67⁺/Ki67⁺ cells, which represent the proportion of cells that were cycling at P7 and remained in proliferation 48h later (Figures 6A and 6B). We observed an increase (from 19.8% to 33.7%) of EdU⁺;Ki67⁺/Ki67⁺ cells in Arhgef7 cKO cerebella compared with controls. This result is consistent with Arhgef7 cKOs having a longer S phase (hence a higher proportion of cells uptaking EdU) and/or a slower cell cycle.

*Arhgef7* cKO GCPs have a longer S phase and/or a slower cell cycle

(A) Immunostaining of sagittal sections of P9 control and *Arhgef7* cKO cerebella stained for Ki67 (green), EdU (green), and counterstained with DAPI (magenta). Control and *Arhgef7* cKO animals received one injection of EdU at P7. Scale bar: 100μm.

(B) Quantification of the percentage of EdU⁺;Ki67⁺ over Ki67⁺ cells in the EGL. Data are represented as mean ± SEM. Number of animals: control n = 4, *Arhgef7* cKO n = 4. Unpaired t test; ∗p < 0.05.

Overall, following the loss of Arhgef7 in GCPs, at P7, we observed a reduction in proliferation in a restricted region of the cerebellum. Since P7 Arhgef7 cKO cerebella display severe hypoplasia, this suggests that the reduction of proliferation due to the loss of Arhgef7 starts prior to P7, likely sometime between E17.5 and P7, and that it is at some point not only restricted to the EGL of the lobule VI and the primary fissure, but rather throughout the EGL of most of the cerebellum. Ultimately, P60 Arhgef7 cKO display severe cerebellar hypoplasia (Figures 2A–2C), suggesting that the defects are cumulative during development, eventually leading to a more dramatic phenotype. Importantly, the P60 phenotype also indicates that the developmental defect that we observed is not simply a development delay, but a defect that is never recovered.

Discussion

Throughout the development of the cerebellum, GCPs undergo a series of events—early proliferation and specification in the RL, tangential migration, second proliferation in the oEGL, initial differentiation in the iEGL, radial migration through the ML, and final differentiation in the IGL—that are tightly regulated in time and space. In this study, we show that Arhgef7 is instrumental to the correct development of the cerebellum. Following the loss of Arhgef7, GCPs showed defect in their proliferation, intercellular organization, differentiation, and tangential and radial migration.

Arhgef7 is important for GCP proliferation and cerebellar morphogenesis

Prior to this study, there was limited evidence connecting Arhgef7 to the regulation of proliferation, particularly within the nervous system.⁴⁰^,⁴¹ Our work shows that loss of Arhgef7 reduces proliferation in postnatal GCPs during cerebellar development. This could explain several of the phenotypes that we observed. First, reduced proliferation could explain the cerebellar hypoplasia we saw in Arhgef7 cKO mice. Second, a previous study has shown that proliferation of GCPs might be the earliest key process in the establishment of anchoring centers, and thus in the foliation and morphogenesis of the cerebellum.⁴² Indeed, they suggest that increased GCP proliferation at the anchoring points provides the driving physical force that starts the invagination of the EGL and the formation of the lobes.⁴² Arhgef7 could thus regulate GCP proliferation, promoting it locally at the anchoring centers, ensuring the correct formation of lobes and ultimately lobules. Overall, our study shows that Arhgef7 is important for the control of proliferation of GCPs during cerebellar development.

Arhgef7 controls GCP organization and migration

One of the first defects to appear in Arhgef7 cKO cerebella is the intercellular disorganization of the EGL at E17.5, resulting in an uneven boundary between the EGL and the ML. This disorganization persists at least until P7 in lobule VI. This defect could be caused by a misregulation of proliferation, either with GCPs proliferating ectopically or proliferating more than they would normally do. However, we observed a reduction, rather than an increase, in GCP proliferation. Moreover, neither Ki67 nor pH3 stainings showed displaced proliferating GCPs outside of the oEGL. Together, this suggests that the EGL disorganization that we observed is not due to a proliferation defect, but rather to a defect in the cellular layering at the border between the EGL and the ML. Calbindin stainings showed no disorganization of the ML, which suggests that the GCPs are responsible for the disorganization. Interestingly, in H&E stainings of P7 control animals, GCPs appear tightly close to each other in the oEGL and looser and more separated in the iEGL. In Arhgef7 cKO cerebella, we observed clusters of loose GCPs in the oEGL as well, and the more protruding pockets of GCPs in the iEGL were always composed of loose GCPs. Moreover, neither proliferating markers (Ki67 and pH3) nor differentiating markers (Tag-1 and GCPs with low levels of NeuN) showed defect in the segregation of proliferating and differentiating GCPs within the EGL. Together, this suggests that Arhgef7 might play a role in the cohesiveness of GCPs: Following the loss of Arhgef7, loose proliferating GCPs might be converted to tight ones inside the oEGL and tight differentiating GCPs to loose differentiating ones in the iEGL with an increased permissiveness to invade the ML. In any case, this defect could lead to the impaired organization of the anchoring centers, as well as an incorrect scaffold for proper division and migration of GCPs, eventually leading to hypoplasia and foliation defects. This would be consistent with one of the most studied contexts of the GIT-Arhgef7 complex, which is focal adhesion, integrin-mediated cell spreading, and cell motility (reviewed by Frank and Hansen⁴³). Disruption of focal adhesions, which serve as physical anchors between cells and the extracellular matrix, could explain the disorganization of the EGL we observed in Arhgef7 cKO cerebella. Consistent with a potential role for GIT proteins with Arhgef7 in this process, scRNA-seq³² analysis showed that GIT1 and GIT2 are also expressed in proliferating and post-mitotic GCPs (Figures S5B and S5C).

Another early defect in Arhgef7 cKO cerebella is the possible reduction in GCP tangential migration at E17.5. In later stages, GCPs appeared to have completed their tangential migration, which suggests that it is only delayed in Arhgef7 cKO mice. This delay could be due to a reduced intrinsic migratory potential of GCPs or an impaired environment for migration due to the disorganization of the EGL. A reduced intrinsic migratory potential would be consistent with one of the classic roles of Arhgef7. Indeed, Arhgef7 germline knockout embryos die due to a migration defect of the endoderm,³³ and Arhgef7 is crucial to propel collective mesoderm migration.⁴⁴ Arhgef7 also negatively regulates focal adhesion maturation and promotes lamellipodial protrusion and focal adhesion turnover, key events required for cell migration.⁴⁵ Also, we cannot rule out the possibility that GCPs do not migrate less, but instead have partially lost the directionality of their migration, and thus are delayed as they are moving randomly inside the EGL rather than perfectly tangential to the cerebellar surface.

Interestingly, following the loss of Arhgef7, the radial migration of GCPs is also impaired. In our EdU labeling experiments, we observed fewer GCPs in the IGL of the Arhgef7 cKO cerebella, suggesting that GCPs are migrating slower or are delayed in their migration. Also, in the ML of Arhgef7 cKO cerebella, we observed fewer GCPs with an elongated soma along the radial axis, which is a characteristic of the radially migrating GCPs. Notably, Arhgef7 cKO cerebella displayed a temporary accumulation of NeuN⁺ cells in the iEGL, a result also consistent with a migration defect and/or a slower differentiation leading to a delayed migration. Overall, this shows that Arhgef7 is important for the radial migration of GCPs as well as the tangential migration. Moreover, it is unclear how this defect in radial migration eventually impacts cerebellar development, especially cerebellar size, as all GCPs eventually leave the EGL since the layer is no longer observable in adult cerebella of both control and Arhgef7 cKO animals. A possibility is that the GCPs that do not engage in their radial migration or are too late to do so die of apoptosis at a stage later than the stage that we studied, similarly to what is observed in GCPs lacking Rac1, which accumulate in the EGL and die of apoptosis before being able to radially migrate.¹⁹

Signaling upstream of Arhgef7

An intriguing question emerging from our study is: through which signaling pathway(s) does Arhgef7 operate during cerebellar development? Among the extrinsic factors regulating GCP development, studies have highlighted the importance of secreted axon guidance cues, including Netrin-1 and its receptor Deleted in Colorectal Cancer (DCC). Netrin-1/DCC seems to have a complex role in GCPs during cerebellar development, possibly playing a role of attractant or repellant, depending on the developmental step GCPs undergo and the precise set of receptors they express at their cell surface.¹⁷^,⁴⁶^,⁴⁷ A recent study demonstrated that Arhgef7 is downstream of Netrin-1/DCC signaling in the attraction of commissural axons in the developing spinal cord.³¹ Thus, Arhgef7 could regulate cerebellar development downstream of Netrin-1/DCC.

Only a few studies have linked Netrin-1 to proliferation, and not through DCC but through Unc5B,⁴⁸^,⁴⁹ another Netrin-1 receptor, and not in neurons. It is thus unlikely that Netrin-1/DCC signaling could explain the reduction in proliferation we observed in Arhgef7 cKO cerebella. Traditionally, Netrin-1/DCC signaling is known for its role during axon guidance⁵⁰^,⁵¹ and also cell survival through dependence receptors.⁵² However, we did not observe increased apoptosis following the loss of Arhgef7.

Netrin-1/DCC is instrumental to the establishment of domains and their boundaries in the developing spinal cord,⁵⁰^,⁵³^,⁵⁴ and many studies have linked Netrin-1 and DCC to migration.⁴⁹^,⁵⁵^,⁵⁶^,⁵⁷^,⁵⁸ Interestingly, Netrin-1, DCC, and Unc5c—another Netrin-1 receptor also involved in neural migration⁵⁹^,⁶⁰^,⁶¹—have been implicated in the radial migration of GCPs during cerebellar development.¹⁷ The precise regulation of DCC and Unc5c at the cell surface modulates GCP response to Netrin-1 and controls their exit out of the EGL, i.e., the time they remain inside the iEGL, and their radial migration.¹⁷ Given our observation that NeuN⁺ GCPs transiently accumulate in the iEGL in Arhgef7 cKOs, it is possible that the disorganization and the migration defects we observed in Arhgef7 cKO GCPs are due to a defect in Netrin-1/DCC signaling. Consistent with a potential role for Netrin-1, Dcc, and Unc5c together with Arhgef7 in GCPs, we observed using scRNA-seq³² that Netrin-1 is expressed in proliferating GCPs and that Dcc and Unc5c are expressed in proliferating and post-mitotic GCPs (Figures S5D–S5F).

Signaling downstream of Arhgef7

Arhgef7 is a RhoGEF that functions as an activator of Rac1 and Cdc42. In previous studies, Rac1 and Cdc42 have been inactivated in GCPs during cerebellar development using GFAP-Cre¹⁹ and Math1-Cre,²⁰ respectively.

Following Rac1 deletion, cerebella displayed severe hypoplasia.¹⁹ In addition, the EGL thickness was increased (which is a defect we did not observe in Arhgef7 cKO cerebella), notably due to a defect in radial migration. These GCPs died of apoptosis inside the EGL before they were able to migrate. No defect in proliferation was observed. Overall, although the cerebellar hypoplasia is similar, the phenotypes following Rac1 and Arhgef7 deletion are vastly different. It is thus unlikely that Rac1 is a major effector of Arhgef7 in GCPs.

In a similar manner, following Cdc42 deletion, cerebella displayed severe hypoplasia.²⁰ At P7, the morphology of the Cdc42 cKO cerebella looked similar to the Arhgef7 cKO cerebella, notably in the VI/VII lobules area. No defect in proliferation or apoptosis was observed. Interestingly, radially migrating GCPs did not have the characteristic elongated soma and were instead more irregular. This is reminiscent of the observation we made in Arhgef7 cKO cerebella, in which we noted an almost complete absence of these elongated somas. It is thus possible that Arhgef7 controls cerebellar development, or at least the radial migration of GCPs, through the activation of Cdc42 in GCPs.

In summary, our work shows that Arhgef7 plays an important role in cerebellum development. Interestingly, we observed various degrees of defects in different cerebellar lobules. This might reflect the differences in developmental timing between each lobule, or possibly that the GCP population is more heterogeneous than what is usually thought. Deciphering the nuances of GCPs in time and space might be key to better understand the cerebellum development.

Limitations of the study

Arhgef7 mutant GCPs show an increase in their EdU⁺;Ki67⁺/Ki67⁺ GCP ratio. Two non-exclusive reasons might explain this result: (i) There might be proportionally more cells in S phase in the Arhgef7 mutant at the time of EdU injection (due, for example, to a slower S phase in the mutant cells), and thus more cells remain labeled 48h later; or (ii) the Arhgef7 mutant GCPs might be cycling more slowly, and thus more GCPs that received the EdU at P7 remain in proliferation 48h later. While a slower S phase and/or a longer time before exiting the cell cycle might explain these results, more experiments will be needed to fully assess the detailed impact of loss of Arhgef7 on GCP cell cycle kinetics, such as EdU/BrdU double labeling experiments.

Another interesting point is that, while the overall proportion of NeuN⁺ cells in Arhgef7 mutant versus control GCPs remains unchanged, their spatial distribution is altered. Specifically, a higher number of NeuN⁺ cells are observed in the EGL of Arhgef7 mutants compared with controls. Two non-exclusive possibilities might explain this result: (i) There might be more NeuN⁺ cells temporarily lingering in the EGL due to a migration defect or (ii) loss of Arhgef7 might slow down the differentiation of GCPs within the iEGL, leading to their accumulation until they finish differentiation and eventually migrate to the ML and IGL. Our EdU pulse/chase experiments support both possibilities: slower migration and/or slower differentiation causing a slower migration. Additional experiments such as timelapse imaging will be useful in differentiating between a slower differentiation versus an impairment in their intrinsic migration ability.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Frédéric Charron (frederic.charron@ircm.qc.ca).

Materials availability

All reagents generated in this study are available upon request to the lead contact.

Data and code availability

•
This paper analyzes existing, publicly available data from Vladoiu et al.³² (GSE118068).
•
This paper does not report original code.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

We thank Kevin Zhang for technical assistance. This study was funded by the Canadian Institutes of Health Research (CIHR) grants PJT-173307, PJT-180637, and PJT-180647 and the Canada Foundation for Innovation grants CFI33768 and CFI NeuroBasis 39794. V.J.-A. received postdoctoral fellowships from CIHR and Fonds de recherche du Québec–Santé. F.C. holds the Canada Research Chairs in Developmental Neurobiology.

Author contributions

Conceptualization, V.J.-A. and F.C.; methodology, V.J.-A., N.B., S.S., P.T.Y., and F.C.; investigation, V.J.-A.; writing - original draft, H.D. and F.C.; writing - review and editing, H.D., P.T.Y., and F.C.; funding acquisition, F.C.; supervision, F.C.

Declaration of interests

Authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Rabbit polyclonal anti-cleaved Caspase-3 (Asp175) (1:500 for IF)	Cell Signaling Technology	Cat#: 9661; RRID: AB_2341188
Rabbit monoclonal anti-Ki67 [SP6] (1:1000 for IF)	Abcam	Cat#: ab16667; RRID: AB_302459
Goat polyclonal anti-Contactin-2/TAG1 (1:400 for IF)	R&D Systems	Cat#: AF4439; RRID: AB_2044647
Rabbit polyclonal anti-phospho-Histone H3 (Ser10) (1:500 for IF)	Millipore	Cat#: 06–570; RRID: AB_310177
Rabbit polyclonal anti-betaPix/Arhgef7 (SH3 domain) (1:200 for IF)	Millipore	Cat#: 07–1450; RRID: AB_11212273
Rabbit monoclonal anti-NeuN (Alexa Fluor® 647 conjugated) (1:1000)	Abcam	Cat#: ab190565; RRID: AB_2732785
Mouse monoclonal anti-Calbindin (clone L109/57) (1:50 for IF)	DSHB	Cat# L109/57; RRID: AB_2877197
Donkey anti-mouse IgG-Alexa Fluor 488	Jackson ImmunoResearch	Cat#: 715-545-151
Donkey anti-mouse IgG-Cy3 conjugated affinity pure	Jackson ImmunoResearch	Cat#: 715-165-151
Donkey anti-goat IgG-Alexa Fluor 488	Molecular Probes	Cat#: A11055
Donkey anti-rabbit IgG-Alexa Fluor 488	Molecular Probes	Cat#: A21206
Donkey anti-rabbit IgG-Alexa Fluor 594	Molecular Probes	Cat#: A21207
Donkey anti-rabbit-IgG-Alex Fluor 647	Jackson ImmunoResearch	Cat#: 711-605-152

Chemicals, peptides, and recombinant proteins

5-Ethynyl-2′-deoxyuridine (EdU)	Sigma	Cat#: 900584
L-Ascorbic acid (vitamin C)	Sigma	Cat#: A4403
Copper(II) sulfate pentahydrate (CuSO₄.5H₂O)	Sigma	Cat#: 939315
Alexa Fluor™ 488 Azide	Invitrogen	Cat#: A10266
DAPI	Sigma-Aldrich	Cat#: D95964
Mayer’s Hematoxylin	Sigma	Cat#: MHS32
Eosin Y (Vintage)	StatLab	Cat#: SL101
Xylenes (certified ACS)	ThermoFisher	Cat#: X5-4
Epredia ClearVue Mountants	ThermoFisher	Cat#: 23-425-401
Bovine serum albumin (BSA)	MultiCell	Cat#: 500-0206
Donkey serum	Wisent	Cat#: 035-150
Mowiol 4-88	Sigma-Aldrich	Cat#: 81381

Deposited data

scRNA-seq dataset of embryonic and postnatal (from E10 to P14) cerebellum development	Vladoiu et al.³²	GSE118068

Experimental models: Organisms/strains

Mouse C57BL/6	The Jackson Laboratory	RRID: IMSR_JAX:000664
Mouse B6.Cg-Tg (Atoh1-cre)1Bfri/J	The Jackson Laboratory (Matei, Pauley et al.³⁴)	RRID: IMSR_JAX:011104
Mouse Arhgef7 tm1a(eucommWtsi) null allele	Kind gift from Tatiana Omelchenko and Kathryn V Anderson (Omelchenko, Rabadan et al.³³)	N/A
Mouse Arhgef7 tm1c(eucommWtsi) conditional allele	Kind gift from Tatiana Omelchenko and Kathryn Anderson (Omelchenko, Rabadan et al.³³)	N/A

Software and algorithms

Prism 8 for Windows	Graphpad	https://www.graphpad.com
ImageJ/FIJI	NIH	https://imagej.net
BBrowserX	BioTuring	https://bioturing.com/single-cell-analysis-bbrowserx
Imaris	Oxford Instruments	https://imaris.oxinst.com

Open in a new tab

Experimental model and study participant details

Mouse lines

All animal work in this study was performed according to the Canadian Council on Animal Care guidelines. The animal protocols 2020-02-FC / 2024-04-FC were approved by the Montreal Clinical Research Institute Animal Care Committee. All mice were maintained in the IRCM specific pathogen-free animal facility in static microinsulator cages, with up to five mice per cage at a temperature of 20°C–24°C and 40–70% humidity. All mice were maintained on a C57BL/6 (The Jackson Laboratory RRID :IMSR_JAX:000664) background. Mice harboring the Arhgef7tm1b(eucommWtsi) null allele and Arhgef7tm1c(eucommWtsi) conditional allele were kindly provided by T. Omelchenko and K. V. Anderson.³³ The Math1-Cre mice line has been previously described.³⁴ E17.5 embryos, P7 mice, P7 mice, and P60 mice of both sexes (not determined) were randomly used for the experiments. Thus, we do not know if sex might influence the results of our study.

Method details

Histology, immunohistochemistry, and immunofluorescence

For staining of cerebellar sections, adult animals were anesthetized using Ketamine/Xylazine administrated by intraperitoneal (IP) injection and perfused with saline (0.9% NaCl) and then with 4% paraformaldehyde (PFA). Whole cerebella were dissected from the adult mice after perfusion. Whole cerebella were also dissected from P7 pups. Embryos were harvested at E15.5 or E17.5. Embryos and dissected cerebella were then fixed (embryos and P7) or postfixed (adult) with 4% PFA for 24 hours, and cryoprotected with 30% sucrose. 12 μm sagittal sections were cut from the frozen tissues and then either stained with hematoxylin and eosin (H&E), or immunostained. Immunostaining was performed by permeabilizing the cerebellar sections with 0.3% Triton X-100 for 15 min followed by washing in PBS, then blocking them with 5% donkey serum with 0.05% Triton X-100 in PBS for 1 h. The blocking solution was replaced with the primary antibody diluted in PBS with 1% donkey serum and 0.05% Triton X-100 and incubated overnight at 4°C. After washing, the samples were incubated with the secondary antibodies diluted in PBS with 1% donkey serum and 0.05% Triton X-100 for 1 h at room temperature. The samples were mounted in Mowiol.

Image acquisition

Image acquisition was performed on either an LSM 700 confocal microscope (Zeiss) or a DM6 microscope (Leica) for fluorescent immunostainings, and on a DM4000B upright microscope (Leica) for H&E stainings.

Antibodies

The following antibodies were used: anti-cleaved caspase-3 (Cell Signaling, 9661S; 1:500 for IF on tissues), anti-Ki67 antibody [SP6] (Abcam, ab16667, 1:500-1000 for IF on tissues), anti-Tag-1 antibody (R&D Systems, AF4439; 1:400 for IF on tissues), anti-phospho-histone H3 (Ser10) antibody (Millipore, 06-570; 1:500 for IF on tissues), anti-Beta-Pix/Arhgef7 (SH3 domain) antibody (Millipore, 07-1450-I; 1:200 for IF on tissues), anti-NeuN (Alexa Fluor 647 conjugated) antibody (Abcam, ab190565, 1:1000 for IF on tissues), anti-Calbindin (clone L109/57) antibody (DSHB, L109/57, 1:50 for IF on tissues).

Cerebellar granule cell precursor migration assay

To assess GCP migration, P7 mice were intraperitoneally injected with EdU (5-Ethynyl-2′-deoxyuridine; 50 mg/kg) and tissues were collected 48h later. EdU incorporation was then revealed by Click-it reaction, using vitamin C (Sigma, A4403; 10 mM), Cu₂SO₄ (Sigma, 939315; 2 mM), and Azide dye (0.5 μM, Invitrogen A10266).

scRNA-seq analysis

We used the scRNA-seq dataset of embryonic and postnatal (from E10 to P14) cerebellum development from Vladoiu et al.³² (GSE118068). Analysis was performed using BBrowserX (BioTuring). We removed blood and vascular cells, leaving 57,928 cells. Uniform Manifold Approximation and Projection (UMAP) projection with a n_neighbors = 30 was used. Cell type annotation was done manually using markers from Vladoiu et al.³² Ki67 corresponds to the mki67 gene and Tag-1 to cntn2.

Quantification and statistical analysis

Quantifications were performed using ImageJ/FIJI (NIH) and Imaris (Oxford Instruments).

Information on statistical tests used, numbers of samples, and statistical measures displayed in the graphs is provided in the figure captions. Error bars represent SEM. Unless otherwise indicated, every experiment was replicated three or more times. No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. When possible, the investigators were blinded to allocation during the experiments and outcome assessment. Statistical tests were performed using Prism (GraphPad).

Published: December 30, 2025

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2025.114580.

Supplemental information

Document S1. Figures S1–S6

mmc1.pdf^{(10.3MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S6

mmc1.pdf^{(10.3MB, pdf)}

Data Availability Statement

•
This paper analyzes existing, publicly available data from Vladoiu et al.³² (GSE118068).
•
This paper does not report original code.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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PERMALINK

Arhgef7 is essential for granule cell precursor proliferation and migration during cerebellum development

Vanesa Jiménez-Amilburu

Hugo Ducuing

Nursen Balekoglu

Sneha Sukumaran

Patricia T Yam

Frédéric Charron

Summary

Graphical abstract

Highlights

Introduction

Results

Arhgef7 is expressed by GCPs during cerebellar development

Figure 1.

Arhgef7 cKO mice display severe hypoplasia and morphogenesis defects in the cerebellum

Figure 2.

Arhgef7 cKO mice have a disorganized EGL architecture

Figure 3.

Loss of Arhgef7 might disrupt embryonic tangential migration of GCPs

Figure 4.

Loss of Arhgef7 impairs postnatal radial migration of GCPs

Arhgef7 cKO cerebella display reduced postnatal proliferation, potentially delayed differentiation, and no increase in apoptosis

Figure 5.

Figure 6.

Discussion

Arhgef7 is important for GCP proliferation and cerebellar morphogenesis

Arhgef7 controls GCP organization and migration

Signaling upstream of Arhgef7

Signaling downstream of Arhgef7

Limitations of the study

Resource availability

Lead contact

Materials availability

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

STAR★Methods

Key resources table

Experimental model and study participant details

Mouse lines

Method details

Histology, immunohistochemistry, and immunofluorescence

Image acquisition

Antibodies

Cerebellar granule cell precursor migration assay

scRNA-seq analysis

Quantification and statistical analysis

Footnotes

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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